Regulation of Rab8 in Toll-Like Receptor Signalling Pathways

Small GTPases of the Rab family have wide ranging and essential roles in recruiting effectors to mediate membrane trafficking and receptor signalling events in mammalian cells. Nucleotide loading and activation/deactivation of the Rabs themselves are regulated by guanine nucleotide exchange factors (GEFs), GTPase activating proteins (GAPs) and other accessory proteins. While Rabs have important roles in innate immune cells, for host-pathogen interactions, receptor signalling and membrane trafficking, in many cases their GEFs and other accessory proteins have not been elucidated.Pathogen-activated, Toll-like receptors (TLRs) initiate and modulate transcription of inflammatory cytokines and other innate immune responses which are important for immune defence but can also contribute to chronic disease. Macrophages are key cells of the innate immune system and previous work from our laboratory established a role for promiscuous Rab family member, Rab8a, in TLR signalling. Rab8a is activated in TLR pathways to recruit phosphoinositide 3-kinase gamma (PI3Kg) as an effector which upregulates TLR-induced Akt/mTOR signalling to drive a biased program of anti-inflammatory cytokines. TLR-associated PI3Kg has now emerged as a key mediator of macrophage programming (or polarisation) in inflammation and cancer and its cognate GTPase, Rab8a, has a prominent role in immunity and disease. Rab8a is itself recruited to macrophage ruffles and macropinosomes through its association with the TLR crosstalk-activated endocytic receptor LRP1. However, it is not yet known how Rab8a is activated in TLR pathways. This project set out to identify the essential Rab8a GEF(s) needed to activate Rab8a as part of the LRP1 complex.Two well-known Rab8 GEFs, GRAB and Rabin8, which previously were uncharacterised in macrophages, were investigated in this project. GRAB was identified as part of the low density lipoprotein receptor-related protein 1 (LRP1) complex in pull-downs analysed by mass spectrometry. Co-immunoprecipitation and fluorescence microscopy showed that both GRAB and the structurally similar GEF, Rabin8, undergo LPS-inducible binding to Rab8a and are localised at sites of Rab8a enrichment. To carry out functional studies, stable knockouts (KOs) of Rabin8, GRAB, as well as a double KO were produced via CRISPR-Cas9 gene editing in macrophage cell lines. Nucleotide activation assays were developed for this project and live-cell imaging with KO cell lines showed that both GEFs contribute additively to TLR4-induced Rab8a GTP-loading, but they are not needed for Rab8a membrane recruitment. Analysis of TLR signalling after double KO of both GEFs suggested redundant roles for Rabin8 and GRAB in activating Rab8a for PI3Kg-dependent Akt/mTOR signalling. Live cell imaging utilising a fluorescent Akt1 reporter confirmed that LPS/TLR-induced Akt signalling is generated on macropinosomes and requires GRAB and Rabin8 GEF function.Next, to investigate possible regulators of the Rab8 GEFs, pull-downs and mass spectrometry were performed and identified a known multi-Rab effector, oculocerebrorenal syndrome of Lowe (OCRL) as a Rabin8 binding protein in LPS activated macrophages. Follow-up experiments provided initial characterisation of a novel OCRL-Rabin8 interaction and indicate a possible recruitment mechanism for the Rab8 GEF during TLR signalling.In conclusion, these results identified both GRAB and Rabin8 as essential activators of Rab8 downstream of TLR4 for inflammatory signalling in mouse macrophages. The results contribute these GEFs and other possible Rab recruiters to an expanding molecular complex that drives PI3Kg/Akt/mTOR signalling for control of inflammation and innate immune responses

Search for dark matter produced in association with bottom or top quarks in √s = 13 TeV pp collisions with the ATLAS detector

A search for weakly interacting massive particle dark matter produced in association with bottom or top quarks is presented. Final states containing third-generation quarks and miss- ing transverse momentum are considered. The analysis uses 36.1 fb−1 of proton–proton collision data recorded by the ATLAS experiment at √s = 13 TeV in 2015 and 2016. No significant excess of events above the estimated backgrounds is observed. The results are in- terpreted in the framework of simplified models of spin-0 dark-matter mediators. For colour- neutral spin-0 mediators produced in association with top quarks and decaying into a pair of dark-matter particles, mediator masses below 50 GeV are excluded assuming a dark-matter candidate mass of 1 GeV and unitary couplings. For scalar and pseudoscalar mediators produced in association with bottom quarks, the search sets limits on the production cross- section of 300 times the predicted rate for mediators with masses between 10 and 50 GeV and assuming a dark-matter mass of 1 GeV and unitary coupling. Constraints on colour- charged scalar simplified models are also presented. Assuming a dark-matter particle mass of 35 GeV, mediator particles with mass below 1.1 TeV are excluded for couplings yielding a dark-matter relic density consistent with measurements

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

Impact of COVID-19 on cardiovascular testing in the United States versus the rest of the world

Objectives: This study sought to quantify and compare the decline in volumes of cardiovascular procedures between the United States and non-US institutions during the early phase of the coronavirus disease-2019 (COVID-19) pandemic. Background: The COVID-19 pandemic has disrupted the care of many non-COVID-19 illnesses. Reductions in diagnostic cardiovascular testing around the world have led to concerns over the implications of reduced testing for cardiovascular disease (CVD) morbidity and mortality. Methods: Data were submitted to the INCAPS-COVID (International Atomic Energy Agency Non-Invasive Cardiology Protocols Study of COVID-19), a multinational registry comprising 909 institutions in 108 countries (including 155 facilities in 40 U.S. states), assessing the impact of the COVID-19 pandemic on volumes of diagnostic cardiovascular procedures. Data were obtained for April 2020 and compared with volumes of baseline procedures from March 2019. We compared laboratory characteristics, practices, and procedure volumes between U.S. and non-U.S. facilities and between U.S. geographic regions and identified factors associated with volume reduction in the United States. Results: Reductions in the volumes of procedures in the United States were similar to those in non-U.S. facilities (68% vs. 63%, respectively; p = 0.237), although U.S. facilities reported greater reductions in invasive coronary angiography (69% vs. 53%, respectively; p < 0.001). Significantly more U.S. facilities reported increased use of telehealth and patient screening measures than non-U.S. facilities, such as temperature checks, symptom screenings, and COVID-19 testing. Reductions in volumes of procedures differed between U.S. regions, with larger declines observed in the Northeast (76%) and Midwest (74%) than in the South (62%) and West (44%). Prevalence of COVID-19, staff redeployments, outpatient centers, and urban centers were associated with greater reductions in volume in U.S. facilities in a multivariable analysis. Conclusions: We observed marked reductions in U.S. cardiovascular testing in the early phase of the pandemic and significant variability between U.S. regions. The association between reductions of volumes and COVID-19 prevalence in the United States highlighted the need for proactive efforts to maintain access to cardiovascular testing in areas most affected by outbreaks of COVID-19 infection

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival