Puromycin-based vectors promote a ROS-dependent recruitment of PML to nuclear inclusions enriched with HSP70 and Proteasomes

<p>Abstract</p> <p>Background</p> <p>Promyelocytic Leukemia (PML) protein can interact with a multitude of cellular factors and has been implicated in the regulation of various processes, including protein sequestration, cell cycle regulation and DNA damage responses. Previous studies reported that misfolded proteins or proteins containing polyglutamine tracts form aggregates with PML, chaperones, and components of the proteasome, supporting a role for PML in misfolded protein degradation.</p> <p>Results</p> <p>In the current study, we have identified a reactive oxygen species (ROS) dependent aggregation of PML, small ubiquitin-like modifier 1 (SUMO-1), heat shock protein 70 (HSP70) and 20S proteasomes in human cell lines that have been transiently transfected with vectors expressing the puromycin resistance gene, puromycin n-acetyl transferase (pac). Immunofluorescent studies demonstrated that PML, SUMO-1, HSP70 and 20S proteasomes aggregated to form nuclear inclusions in multiple cell lines transfected with vectors expressing puromycin (puro) resistance in regions distinct from nucleoli. This effect does not occur in cells transfected with identical vectors expressing other antibiotic resistance genes or with vectors from which the pac sequence has been deleted. Furthermore, ROS scavengers were shown to ablate the effect of puro vectors on protein aggregation in transfected cells demonstrating a dependency of this effect on the redox state of transfected cells.</p> <p>Conclusion</p> <p>Taken together we propose that puromycin vectors may elicit an unexpected misfolded protein response, associated with the formation of nuclear aggresome like structures in human cell lines. This effect has broad implications for cellular behavior and experimental design.</p

Obesity in adults: a 2022 adapted clinical practice guideline for Ireland

This Clinical Practice Guideline (CPG) for the management of obesity in adults in Ireland, adapted from the Canadian CPG, defines obesity as a complex chronic disease characterised by excess or dysfunctional adiposity that impairs health. The guideline reflects substantial advances in the understanding of the determinants, pathophysiology, assessment, and treatment of obesity. It shifts the focus of obesity management toward improving patient-centred health outcomes, functional outcomes, and social and economic participation, rather than weight loss alone. It gives recommendations for care that are underpinned by evidence-based principles of chronic disease management; validate patients' lived experiences; move beyond simplistic approaches of "eat less, move more" and address the root drivers of obesity. People living with obesity face substantial bias and stigma, which contribute to increased morbidity and mortality independent of body weight. Education is needed for all healthcare professionals in Ireland to address the gap in skills, increase knowledge of evidence-based practice, and eliminate bias and stigma in healthcare settings. We call for people living with obesity in Ireland to have access to evidence-informed care, including medical, medical nutrition therapy, physical activity and physical rehabilitation interventions, psychological interventions, pharmacotherapy, and bariatric surgery. This can be best achieved by resourcing and fully implementing the Model of Care for the Management of Adult Overweight and Obesity. To address health inequalities, we also call for the inclusion of obesity in the Structured Chronic Disease Management Programme and for pharmacotherapy reimbursement, to ensure equal access to treatment based on health-need rather than ability to pay

Myth or Memory? Recollections of Penal Times in Irish Folklore

Stories of priests being hunted down and murdered at Mass Rocks by priest catchers and soldiers during the Penal era in Ireland persist to the present day. Using Ó Ciosáin’s (2004) tripartite taxonomy of memory this paper explores the reasons why these images continue to dominate and reflect persecuted nature of Catholicism

Identification and Expression of Multidrug Transporters Responsible for Fluconazole Resistance in Candida dubliniensis

Candida dubliniensis is a recently described Candida species associated with oral candidosis in human immunodeficiency virus (HIV)-infected and AIDS patients, from whom fluconazole-resistant clinical isolates have been previously recovered. Furthermore, derivatives exhibiting a stable fluconazole-resistant phenotype have been readily generated in vitro from fluconazole-susceptible isolates following exposure to the drug. In this study, fluconazole-resistant isolates accumulated up to 80% less [(3)H]fluconazole than susceptible isolates and also exhibited reduced susceptibility to the metabolic inhibitors 4-nitroquinoline-N-oxide and methotrexate. These findings suggested that C. dubliniensis may encode multidrug transporters similar to those encoded by the C. albicans MDR1, CDR1, and CDR2 genes (CaMDR1, CaCDR1, and CaCDR2, respectively). A C. dubliniensis homolog of CaMDR1, termed CdMDR1, was cloned; its nucleotide sequence was found to be 92% identical to the corresponding CaMDR1 sequence, while the predicted CdMDR1 protein was found to be 96% identical to the corresponding CaMDR1 protein. By PCR, C. dubliniensis was also found to encode homologs of CDR1 and CDR2, termed CdCDR1 and CdCDR2, respectively. Expression of CdMDR1 in a fluconazole-susceptible Δpdr5 null mutant of Saccharomyces cerevisiae conferred a fluconazole-resistant phenotype and resulted in a 75% decrease in accumulation of [(3)H]fluconazole. Northern analysis of fluconazole-susceptible and -resistant isolates of C. dubliniensis revealed that fluconazole resistance was associated with increased expression of CdMDR1 mRNA. In contrast, most studies showed that overexpression of CaCDR1 was associated with fluconazole resistance in C. albicans. Increased levels of the CdMdr1p protein were also detected in fluconazole-resistant isolates. Similar results were obtained with fluconazole-resistant derivatives of C. dubliniensis generated in vitro, some of which also exhibited increased levels of CdCDR1 mRNA and CdCdr1p protein. These results demonstrate that C. dubliniensis encodes multidrug transporters which mediate fluconazole resistance in clinical isolates and which can be rapidly mobilized, at least in vitro, on exposure to fluconazole

Endoglin-Mediated Suppression of Prostate Cancer Invasion Is Regulated by Activin and Bone Morphogenetic Protein Type II Receptors

<div><p>Mortality from prostate cancer (PCa) is due to the formation of metastatic disease. Understanding how that process is regulated is therefore critical. We previously demonstrated that endoglin, a type III transforming growth factor β (TGFβ) superfamily receptor, suppresses human PCa cell invasion and metastasis. Endoglin-mediated suppression of invasion was also shown by us to be dependent upon the type I TGFβ receptor, activin receptor-like kinase 2 (ALK2), and the downstream effector, Smad1. In this study we demonstrate for the first time that two type II TGFβ receptors are required for endoglin-mediated suppression of invasion: activin A receptor type IIA (ActRIIA) and bone morphogenetic protein receptor type II (BMPRII). Downstream signaling through these receptors is predominantly mediated by Smad1. ActRIIA stimulates Smad1 activation in a kinase-dependent manner, and this is required for suppression of invasion. In contrast BMPRII regulates Smad1 in a biphasic manner, promoting Smad1 signaling through its kinase domain but suppressing it through its cytoplasmic tail. BMPRII’s Smad1-regulatory effects are dependent upon its expression level. Further, its ability to suppress invasion is independent of either kinase function or tail domain. We demonstrate that ActRIIA and BMPRII physically interact, and that each also interacts with endoglin. The current findings demonstrate that both BMPRII and ActRIIA are necessary for endoglin-mediated suppression of human PCa cell invasion, that they have differential effects on Smad1 signaling, that they make separate contributions to regulation of invasion, and that they functionally and physically interact.</p></div

ActRIIA promotes Smad1 signaling while BMPRII is inhibitory.

<p>PC3-M cells were transiently transfected with empty vector or endoglin and the indicated siRNA as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072407#pone-0072407-g001" target="_blank">Figure 1</a>. Two days later cells were lysed for Western blot (<b>A</b>) or luciferase promoter assay (<b>B</b>). <b>A</b>) ActRIIA and BMPRII differentially regulate Smad1 protein phosphorylation. Western blot on resultant cell lysate was performed for Smad1, phospho-Smad1/5 (pSmad1/5), endoglin and GAPDH. Data are from a representative experiment (N = 4 experiments). <b>B</b>) ActRIIA and BMPRII differentially regulate BRE₂-luciferase activation. Cells were additionally co-transfected with BRE₂-luciferase and <i>Renilla</i> luciferase constructs, and luciferase activity (normalized to <i>Renilla</i> luciferase activity) was measured. Data are the mean ± SD from a single experiment conducted in replicates of N = 2, conducted three separate times with similar results (also N = 2). *, p≤0.05 between the indicated groups. <b>C</b>) BMP7- and BMP9-stimulated Smad1 phosphorylation is differentially regulated by ActRII and BMPRII. Cells were transfected as above, serum-starved, and treated with BMP7 or BMP9 as indicated. Western blot on resultant cell lysate was performed for phospho-Smad1/5 (pSmad1/5) and total Smad1. Data are from a representative experiment (N = 2 experiments).</p

Smad1 is the main downstream target of endoglin.

<p>PC3-M cells were transfected with endoglin, vector (Vec), or with siRNA to Smad1, (siSm1), Smad5 (siSm5), Smad8 (siSm8), BMPRII (siRII) or non-targeting (siNeg), and processed 48 hrs later as indicated. <b>A</b>) Smad-targeting siRNA suppresses transcript in a Smad-specific fashion. Smad1, -5, and -8 mRNA expression was assessed via qRT-PCR, normalized to GAPDH, and expressed relative to siNeg-transfected cells (normalized to 1.0). Data represent mean ± SD from a single experiment conducted in replicates of N = 2, that was repeated 3 separate times (also in replicates of N = 2) with similar results. *, p≤0.05 compared to siNeg. <b>B</b>) Effect of siRNA on phospho-Smad1/5/8, phospho-Smad1/5, and total Smad1 protein levels. Cell lysates were probed by antibody directed towards phospho-Smad1/5/8 (pSmad1/5/8) and total Smad1 protein by Western blot. The non-specific band (*) immediately under the pSmad1/5/8 band (arrow) confirms even loading. Negative control cells (Neg Ctl) were transfected with vector and serum starved overnight. Positive control cells (Pos Ctl) were transfected with endoglin, not serum starved and were treated with TGFβ for 30 min. Separate samples were similarly transfected and treated, and cell lysates were probed for phospho-Smad1/5 (pSmad1/5). Data are from one representative experiment in each case, repeated 3 separate times with similar results. <b>C</b>) Endoglin-mediated BRE₂-luciferase activity is largely mediated by Smad1. Cells were transfected with endoglin and were additionally co-transfected with BRE₂- and <i>Renilla</i> luciferase construct, and luciferase assays performed. Data represent mean ± SD of a single representative experiment conducted in replicates of N = 2, repeated 3 separate times (replicates of N = 2) with similar results. *, p≤0.05 compared to Endoglin/siNeg. <b>D</b>) BMPRII-mediated suppression of BRE₂-luciferase activity is largely mediated by Smad1. Cells were transfected as in (C) with addition of indicated siRNA and luciferase activity as assessed as above. Data represent mean ± SD of a single representative experiment conducted in replicates of N = 2, repeated 2 separate times (replicates of N = 2) with similar results. *, p≤0.05 compared to Endoglin/siNeg/siBMPRII.</p

ActRIIA and BMPRII physically interact

<p>. Coimmunoprecipitation experiments were performed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072407#pone-0072407-g007" target="_blank">Figure 7</a>. In all experiments, cells were transfected, cell surface proteins crosslinked, immunoprecipitation (IP) performed, crosslinks reversed, and Western blot (IB) performed as indicated. In some studies, cells were transfected with FLAG-endoglin as a positive control. Input lysate, post IP lysate, and IP samples are loaded as indicated. (<b>A and B</b>) ActRIIA precipitates with BMPRII. (<b>C and D</b>) ActRIIA kinase domain is dispensable for interaction with BMPRII. (<b>E and F</b>) BMPRII kinase activity and tail domain are dispensable for interaction with BMPRII. All data are from a representative experiment, repeated at least N = 5 separate times.</p