Constraints on Dark Matter from Colliders

We show that colliders can impose strong constraints on models of dark matter, in particular when the dark matter is light. We analyze models where the dark matter is a fermion or scalar interacting with quarks and/or gluons through an effective theory containing higher dimensional operators which represent heavier states that have been integrated out of the effective field theory. We determine bounds from existing Tevatron searches for monojets as well as expected LHC reaches for a discovery. We find that colliders can provide information which is complementary or in some cases even superior to experiments searching for direct detection of dark matter through its scattering with nuclei. In particular, both the Tevatron and the LHC can outperform spin dependent searches by an order of magnitude or better over much of parameter space, and if the dark matter couples mainly to gluons, the LHC can place bounds superior to any spin independent search.Comment: 23 pages, 16 figure

Human cytomegalovirus pUL79 Is an elongation factor of RNA polymerase II for viral gene transcription

In this study, we have identified a unique mechanism in which human cytomegalovirus (HCMV) protein pUL79 acts as an elongation factor to direct cellular RNA polymerase II for viral transcription during late times of infection. We and others previously reported that pUL79 and its homologues are required for viral transcript accumulation after viral DNA synthesis. We hypothesized that pUL79 represented a unique mechanism to regulate viral transcription at late times during HCMV infection. To test this hypothesis, we analyzed the proteome associated with pUL79 during virus infection by mass spectrometry. We identified both cellular transcriptional factors, including multiple RNA polymerase II (RNAP II) subunits, and novel viral transactivators, including pUL87 and pUL95, as protein binding partners of pUL79. Co-immunoprecipitation (co-IP) followed by immunoblot analysis confirmed the pUL79-RNAP II interaction, and this interaction was independent of any other viral proteins. Using a recombinant HCMV virus where pUL79 protein is conditionally regulated by a protein destabilization domain ddFKBP, we showed that this interaction did not alter the total levels of RNAP II or its recruitment to viral late promoters. Furthermore, pUL79 did not alter the phosphorylation profiles of the RNAP II C-terminal domain, which was critical for transcriptional regulation. Rather, a nuclear run-on assay indicated that, in the absence of pUL79, RNAP II failed to elongate and stalled on the viral DNA. pUL79-dependent RNAP II elongation was required for transcription from all three kinetic classes of viral genes (i.e. immediate-early, early, and late) at late times during virus infection. In contrast, host gene transcription during HCMV infection was independent of pUL79. In summary, we have identified a novel viral mechanism by which pUL79, and potentially other viral factors, regulates the rate of RNAP II transcription machinery on viral transcription during late stages of HCMV infection

Quantification of O-glycosylation stoichiometry and dynamics using resolvable mass tags

Mechanistic studies of O-GlcNAc glycosylation have been limited by an inability to monitor the glycosylation stoichiometries of proteins obtained from cells. Here we describe a powerful method to visualize the O-GlcNAc–modified protein subpopulation using resolvable polyethylene glycol mass tags. This approach enables rapid quantification of in vivo glycosylation levels on endogenous proteins without the need for protein purification, advanced instrumentation or expensive radiolabels. In addition, it establishes the glycosylation state (for example, mono-, di-, tri-) of proteins, providing information regarding overall O-GlcNAc site occupancy that cannot be obtained using mass spectrometry. Finally, we apply this strategy to rapidly assess the complex interplay between glycosylation and phosphorylation and discover an unexpected reverse 'yin-yang' relationship on the transcriptional repressor MeCP2 that was undetectable by traditional methods. We anticipate that this mass-tagging strategy will advance our understanding of O-GlcNAc glycosylation, as well as other post-translational modifications and poorly understood glycosylation motifs

HIV-1 Broadly Neutralizing Antibody Extracts Its Epitope from a Kinked gp41 Ectodomain Region on the Viral Membrane

SummaryAlthough rarely elicited during natural human infection, the most broadly neutralizing antibodies (BNAbs) against diverse human immunodeficiency virus (HIV)-1 strains target the membrane-proximal ectodomain region (MPER) of viral gp41. To gain insight into MPER antigenicity, immunogenicity, and viral function, we studied its structure in the lipid environment by a combination of nuclear magnetic resonance (NMR), electron paramagnetic resonance (EPR), and surface plasmon resonance (SPR) techniques. The analyses revealed a tilted N-terminal α helix (aa 664–672) connected via a short hinge to a flat C-terminal helical segment (675–683). This metastable L-shaped structure is immersed in viral membrane and, therefore, less accessible to immune attack. Nonetheless, the 4E10 BNAb extracts buried W672 and F673 after initial encounter with the surface-embedded MPER. The data suggest how BNAbs may perturb tryptophan residue-associated viral fusion involving the mobile N-terminal MPER segment and, given conservation of MPER sequences in HIV-1, HIV-2, and SIV, have important implications for structure-guided vaccine design

PP2ARts1 is a master regulator of pathways that control cell size

Cell size checkpoints ensure that passage through G1 and mitosis occurs only when sufficient growth has occurred. The mechanisms by which these checkpoints work are largely unknown. PP2A associated with the Rts1 regulatory subunit (PP2ARts1) is required for cell size control in budding yeast, but the relevant targets are unknown. In this paper, we used quantitative proteome-wide mass spectrometry to identify proteins controlled by PP2ARts1. This revealed that PP2ARts1 controls the two key checkpoint pathways thought to regulate the cell cycle in response to cell growth. To investigate the role of PP2ARts1 in these pathways, we focused on the Ace2 transcription factor, which is thought to delay cell cycle entry by repressing transcription of the G1 cyclin CLN3. Diverse experiments suggest that PP2ARts1 promotes cell cycle entry by inhibiting the repressor functions of Ace2. We hypothesize that control of Ace2 by PP2ARts1 plays a role in mechanisms that link G1 cyclin accumulation to cell growth

Group IIa secretory phospholipase expression correlates with group IIa secretory phospholipase inhibition–mediated cell death in K-ras mutant lung cancer cells

ObjectiveThere are currently no targeted therapies against lung tumors with oncogenic K-ras mutations that are found in 25% to -40% of lung cancers and are characterized by their resistance to epidermal growth factor receptor inhibitors. The isozyme group IIa secretory phospholipase A2 (sPLA2IIa) is a potential biomarker and regulator of lung cancer cell invasion; however, the relationship between K-ras mutations and sPLA2IIa has yet to be investigated. We hypothesize that sPLA2IIa modulates lung cancer cell growth in K-ras mutant cells and that sPLA2IIa expression in human lung tumors is increased in K-ras mutant tumors.MethodsBaseline sPLA2IIa expression in K-ras mutant lung cancer cell lines (A549, SW1573, H358, H2009) was assessed. Cells were treated with a specific sPLA2IIa inhibitor and evaluated for apoptosis and cell viability. Nuclear factor kappa-b (NF-κB) and extracellular signal-regulated kinase 1/2 activity were detected by Western blot. Human tumor samples were evaluated for sPLA2IIa mRNA expression by quantitative reverse-transcription polymerase chain reaction.ResultsCytotoxicity of sPLA2IIa inhibition correlates with sPLA2IIa expression. Apoptosis in response to sPLA2 inhibition parallels attenuation in NF-κB activity. In addition, sPLA2IIa expression in human tumors correlates with squamous cell pathology and increasing stage of K-ras mutant lung tumors.ConclusionsBaseline sPLA2IIa expression predicts response to sPLA2IIa inhibition in some K-ras mutant lung cancer cells. This finding is independent of p53 mutation status. Furthermore, squamous tumors and advanced-stage K-ras mutant tumors express more sPLA2IIa. These data support a role for sPLA2IIa as a potential global therapeutic target in the treatment of lung cancer

Impact of Lung Function on Exacerbations, Health Care Utilization, and Costs Among Patients with COPD

Objective: To evaluate the impact of lung function, measured as forced expiratory volume in 1 second (FEV1) % predicted, on health care resource utilization and costs among patients with COPD in a real-world US managed-care population. Methods: This observational retrospective cohort study utilized administrative claim data augmented with medical record data. The study population consisted of patients with one or more medical claims for pre- and postbronchodilator spirometry during the intake period (July 1, 2012 to June 30, 2013). The index date was the date of the earliest medical claim for pre- and postbronchodilator spirometry. Spirometry results were abstracted from patients\u27 medical records. Patients were divided into two groups (low FEV1% predicted [\u3c 50%] and high FEV1% predicted [≥ 50%]) based on the 2014 Global Initiative for Chronic Obstructive Lung Disease report. Health care resource utilization and costs were based on the prevalence and number of discrete encounters during the 12-month postindex follow-up period. Costs were adjusted to 2014 US dollars. Results: A total of 754 patients were included (n=297 low FEV1% predicted group, n=457 high FEV1% predicted group). COPD exacerbations were more prevalent in the low FEV1% predicted group compared with the high group during the 12-month pre- (52.5% vs 39.6%) and postindex periods (49.8% vs 36.8%). Mean (standard deviation) follow-up all-cause and COPD-related costs were $27,380 ($38,199) and $15,873 ($29,609) for patients in the low FEV1% predicted group, and $22,075 ($28,108) and $10,174 ($18,521) for patients in the high group. In the multivariable analyses, patients in the low FEV1% predicted group were more likely to have COPD exacerbations and tended to have higher COPD-related costs when compared with patients in the high group. Conclusion: Real-world data demonstrate that patients with COPD who have low FEV1% predicted levels use more COPD medications, have more COPD exacerbations, and incur higher COPD-related health care costs than those with high FEV1% predicted levels

Identifying gene network patterns and associated cellular immune responses in children with or without nut allergy

Background: Although evidence suggests that the immune system plays a key role in the pathophysiology of nut allergy, the precise immunological mechanisms of nut allergy have not been systematically investigated. The aim of the present study was to identify gene network patterns and associated cellular immune responses in children with or without nut allergy. Methods: Transcriptome profiling of whole blood cells was compared between children with and without nut allergy. Three genes were selected to be validated on a larger cohort of samples (n = 86) by reverse transcription-polymerase chain reactions (RT-qPCR). The composition of immune cells was inferred from the transcriptomic data using the CIBERSORTx algorithm. A co-expression network was constructed employing weighted gene co-expression network analysis (WGCNA) on the top 5000 most variable transcripts. The modules were interrogated with pathway analysis tools (InnateDB) and correlated with clinical phenotypes and cellular immune responses. Results: Proportions of neutrophils were positively correlated and CD4+ T-cells and regulatory T-cells (Tregs) were negatively correlated with modules of nut allergy. We also identified 2 upregulated genes, namely Interferon Induced With Helicase C Domain 1 (IFIH1), DNA damage-regulated autophagy modulator 1 (DRAM1) and a downregulated gene Zinc Finger Protein 512B (ZNF512B) as hub genes for nut allergy. Further pathway analysis showed enrichment of type 1 interferon signalling in nut allergy. Conclusions: Our findings suggest that upregulation of type 1 interferon signalling and neutrophil responses and downregulation of CD4+ T-cells and Tregs are features of the pathogenesis of nut allergy

Recombinant human erythropoietin promotes the acquisition of a malignant phenotype in head and neck squamous cell carcinoma cell lines in vitro

<p>Abstract</p> <p>Background</p> <p>Recent studies indicate an increase in tumor progression and recurrence in head and neck squamous cell carcinomas (HNSCC) of cancer patients taking recombinant human erythropoietin (rhEpo) for anemia. This study was undertaken to investigate the potential role of rhEpo in invasion, proliferation, and cisplatin-induced cell death in HNSCC cell lines.</p> <p>Methods</p> <p>The following experiments were performed with two HNSCC cell lines, UMSCC-10B and UMSCC-22B. Presence of EpoR in both cell lines was determined by western blot and quantitative PCR. Colorimetric MTS assays and clonogenic assays were used to study the effect of rhEpo at pharmacologically relevant doses on cell proliferation. Matrigel invasion assays were performed in order to determine effects of exogenous rhEpo on invasive abilities. Clonogenic assays were also used to study potential cytoprotective effects of rhEpo against cisplatin. Immunoblotting was done to analyze the effect of rhEpo on Akt phosphorylation. Finally, MTS and TUNEL assays were performed to test our hypothesis that Akt activation by PI3K was involved in rhEpo-mediated cisplatin resistance.</p> <p>Results</p> <p>HNSCC cell lines were shown to express Epo receptor (EpoR). RhEpo increased invasion 1.8-fold in UMSCC-10B and 2.6-fold in UMSCC-22B compared to control. RhEpo at 10 U/ml increased cell proliferation by 41% and 53% in UMSCC-10B and UMSCC-22B, respectively, and colony formation by 1.5-fold and 1.8-fold. UMSCC-10B treated with cisplatin and exposed to rhEpo at 1 and 10 U/ml resulted in a 1.7-fold and 3.0-fold increase in colony number compared to control, respectively. UMSCC-22B treated with cisplatin and rhEpo at 1 or 10 U/ml resulted in ~2.5-fold increase in colony number. A TUNEL assay demonstrated a 30.5% and 76.5% increase in survival in UMSCC-10B and UMSCC-22B cells, respectively, in cisplatin and rhEpo-treated cells compared to cisplatin alone. MTS assay showed similar cytoprotective effects. Western blot revealed increased phosphorylation of Akt upon exposure of HNSCC cell lines to rhEpo. MTS assay and TUNEL analyses implicate Akt as a likely contributor to regulation of rhEpo-mediated cytoprotection.</p> <p>Conclusions</p> <p>The results demonstrate that, in HNSCC cells expressing functional EpoR, rhEpo promotes invasion, cell proliferation, and induces resistance to cisplatin, which may contribute to tumor progression.</p

BCL6 enables Ph+ acute lymphoblastic leukaemia cells to survive BCR-ABL1 kinase inhibition.

Tyrosine kinase inhibitors (TKIs) are widely used to treat patients with leukaemia driven by BCR-ABL1 (ref. 1) and other oncogenic tyrosine kinases. Recent efforts have focused on developing more potent TKIs that also inhibit mutant tyrosine kinases. However, even effective TKIs typically fail to eradicate leukaemia-initiating cells (LICs), which often cause recurrence of leukaemia after initially successful treatment. Here we report the discovery of a novel mechanism of drug resistance, which is based on protective feedback signalling of leukaemia cells in response to treatment with TKI. We identify BCL6 as a central component of this drug-resistance pathway and demonstrate that targeted inhibition of BCL6 leads to eradication of drug-resistant and leukaemia-initiating subclones