Defining functional cooperation of oncogenic KRAS and rare driver mutations in intrahepatic cholangiocarcinom

Intrahepatic cholangiocarcinoma (iCCA) is an aggressive and lethal malignancy arising from the bile ducts. Patients with iCCA typically present with genetically heterogeneous tumours. There is an exception of a small minority of patients where actionable mutations are present, but the heterogeneity of this cancer has hampered the development of targeted therapeutics as the number of recurrent mutations between patients is limited. The majority of iCCA patients are not amenable to surgery, and standard of care chemotherapies act to extend life and are not considered as a curative treatment. As such, there is a significant requirement to explore the functional genetics of iCCA with the goal of developing more effective treatments and therapies. Deep sequencing studies have identified oncogenic mutations in KRAS in iCCA and these represent one of the most recurrent alterations in this cancer. Therefore I focussed on the role of KRAS mutations and their interactions with loss of function mutations. During this work, I identified and validated in vivo which loss of function mutations were capable of interacting with oncogenic KRASG12D mutations. To do this, I developed and carried out a CRISPR-spCas9 library screen informed by mutations that have been previously identified as pathogenic in patient iCCA. Using this approach, I identified the loss of cytoskeletal signalling protein Neurofibromin 2 (NF2) as a novel driver event in iCCA and that loss of this gene interacts with mutant KRAS to initiate cancer. To investigate the role of NF2 further, I explored the relationship between Nf2-loss, Trp53-loss and mutant KRAS, and I found that Nf2 loss cooperated with Trp53 loss to lead to cancer with a severely accelerated phenotype and increased lethality. Additionally, these tumours were representative of a rare and aggressive sarcomatoid subtype of iCCA. To understand how loss of both Trp53 and Nf2 leads to this aggressive phenotype, I used proteomic analysis of a number of signalling pathways, and RNA sequencing to define which mechanisms are altered and contribute to the aggressive cancer phenotype. These analyses identified the co-activation of Wnt/ β-catenin and Pi3K/AKT signalling as recurrently activated in iCCA. To test whether the aggressive nature of dual Trp53 and Nf2 loss was a result of changes in these signalling pathways, I inhibited them pharmacologically with inhibitors targeting Wnt/β-catenin and Pi3K/AKT signalling. Treatment with both of these inhibitors significantly improved survival of tumour bearing mice. Finally, I established a genetically engineered mouse model of iCCA not reliant on Nf2-loss for its progression to address whether inhibition of Wnt/β-catenin and Pi3K/AKT could represent a wide-spanning therapeutic and determine whether the treatment of a different pathological subtype of iCCA would be with these inhibitors would reduce disease progression. I showed this form of well-differentiated iCCA is also amenable to inhibition with this combination of therapies. These data demonstrate that Nf2 is a rare driver gene of iCCA that acts in a cooperative manner with oncogenic KRAS to accelerate tumourigenesis in vivo. Examination of these tumours highlighted a concurrent Wnt and PI3K signalling signature, and I demonstrate that pharmacological co-inhibition substantially impedes iCCA growth, highlighting the use of Wnt/β-catenin and Pi3K/AKT signalling inhibition as a broad treatment for iCCA patients

In vivo modeling of patient genetic heterogeneity identifies new ways to target cholangiocarcinoma.

L. Boulter was funded by The Wellcome Trust (207793/Z/17/Z), AMMF (2016/108, 2017/115), and Cancer Research UK (C52499/A27948). L. Boulter is also supported by an MRC university grant to the MRC Human Genetics Unit

Dataset associated with "Design and Testing of a Low-Cost Sensor and Sampling Platform for Indoor Air Quality"

These data were collected during a study in which nine prototypes of a low-cost sensor and sampling platform---which was called the "Home Health Box" and was designed to measure concentrations of CO2, CO, NO2, O3, PM2.5, and PM10 in indoor air---were collocated with reference (i.e., research- and regulatory-grade) CO2, CO, NO2, O3, and PM2.5 monitors in the kitchen of a home in Fort Collins, Colorado, USA for one week in October 2020. The files associated with this dataset include: (1) raw CO2 concentrations measured using a LI-COR Biosciences LI-820 CO2 Gas Analyzer (the reference CO2 monitor); (2) raw CO concentrations logged by two TSI Incorporated QTrak 7575-X Indoor Air Quality Monitors with model 982 probes (the reference CO monitors); (3) raw PM2.5 concentrations logged by a ThermoFisher Scientific 1405 Tapered Element Oscillating Microbalance (the reference PM2.5 monitor); (4) the pre- and post- sampling masses of 37-mm diameter polytetrafluoroethylene filters used to sample PM2.5 and PM10 with the nine Home Health Boxes and one Access Sensor Technologies ASPEN box; (5) a log of activities that took place inside the home during the experiment; (6) calibration coefficients provided by the manufacturer (Alphasense) of the low-cost electrochemical CO, NO2, and O3 sensors used in the Home Health Boxes; (7) coefficients of linear mixed calibration models fit to relate CO2 concentrations reported by the low-cost nondispersive infrared (NDIR) sensors used in the Home Health Boxes to reference CO2 concentrations; (8) coefficients of linear mixed calibration models fit to relate data recorded by the low-cost electrochemical CO, NO2, and O3 sensors used in the Home Health Boxes to reference CO, NO2, and O3 concentrations; (9) processed time-series CO2, CO, NO2, O3, and PM2.5 concentration data obtained from the Home Health Boxes as well as the reference monitors; (10) raw NO and NOx concentrations measured using a Thermo Environmental Instruments Model 42C Trace Level Chemiluminescence NO-NO2-NOx Analyzer (the reference NO2 monitor); (11) raw O3 concentrations measured using a Thermo Environmental Instruments Model 49C UV Photometric O3 Analyzer (the reference O3 monitor); (12) all raw data logged by the nine Home Health Boxes; (13) raw data logged by a Access Sensor Technologies ASPEN box installed outside the home.Americans spend most of their time indoors at home, but comprehensive characterization of in-home air pollution is limited by the cost and size of reference-quality monitors. We assembled small "Home Health Boxes" (HHBs) to measure indoor PM2.5, PM10, CO2, CO, NO2, and O3 concentrations using filter samplers and low-cost sensors. Nine HHBs were collocated with reference monitors in the kitchen of an occupied home in Fort Collins, Colorado, USA for 168 h while wildfire smoke impacted local air quality. When HHB data were interpreted using gas sensor manufacturers' calibrations, HHBs and reference monitors (a) categorized the level of each gaseous pollutant similarly (as either low, elevated, or high relative to air quality standards) and (b) both indicated that gas cooking burners were the dominant source of CO and NO2 pollution; however, HHB and reference O3 data were not correlated. When HHB gas sensor data were interpreted using linear mixed calibration models derived via collocation with reference monitors, root-mean-square error decreased for CO2 (from 408 to 58 ppm), CO (645 to 572 ppb), NO2 (22 to 14 ppb), and O3 (21 to 7 ppb); additionally, correlation between HHB and reference O3 data improved (Pearson's r increased from 0.02 to 0.75). Mean 168-h PM2.5 and PM10 concentrations derived from nine filter samples were 19.4 micrograms per cubic meter (6.1% relative standard deviation [RSD]) and 40.1 micrograms per cubic meter (7.6% RSD). The 168-h PM2.5 concentration was overestimated by PMS5003 sensors (median sensor/filter ratio = 1.7) and underestimated slightly by SPS30 sensors (median sensor/filter ratio = 0.91)

Unpacking social-ecological transformations: Conceptual, ethical and methodological insights

Social-ecological systems contribute to environmental change and, in turn, face its corresponding shocks and disturbances. As scholarship on resilience and social-ecological transformations grows, researchers from various disciplines continue to debate how major transitions and environmental change can be anticipated, studied or guided towards just and ethical outcomes. To this end, we apply an interdisciplinary perspective to describing key aspects of social-ecological transformations scholarship around three pressing questions: (1) What features or criteria distinguish transformations from other forms of change?; (2) What are the political, ethical, and normative concerns associated with transformations?; and (3) How can we better track, measure, and evaluate such transformations? Our insights, which emerged from a workshop with early-career interdisciplinary scholars, highlight questions of justice, equity and ethics in transformations research, and suggest that more precise indicators of change, a more explicit understanding of system boundaries, and a dual focus on process and outcomes will help advance our understanding of the social-ecological implications of transformations. We hope that articulating these challenges and recommendations in an interdisciplinary framing will help further the conversation on these critical topics and provide an accessible perspective of key considerations for multidisciplinary scholarship on social-ecological transformations

Screening of wild deer populations for exposure to SARS-CoV-2 in the United Kingdom, 2020-2021.

Following findings in Northern America of SARS-CoV-2 infections in white-tailed deer, there is concern of similar infections in European deer and their potential as reservoirs of SARS-CoV-2 including opportunities for the emergence of new variants. UK deer sera were collected in 2020-2021 from 6 species and a hybrid with 1748 tested using anti-spike and anti-nucleocapsid serology assays. No samples were positive on both assays nor by surrogate neutralization testing. There is no evidence that spill-over infections of SARS-CoV-2 occurred from the human population to UK deer or that SARS-CoV-2 has been circulating in UK deer (over the study period). Although it cannot be ruled out, study results indicate that spill-over infections followed by circulation of SARS-CoV-2 to the most common European deer species is small

CCDC65 mutation causes primary ciliary dyskinesia with normal ultrastructure and hyperkinetic cilia.

BACKGROUND: Primary ciliary dyskinesia (PCD) is a genetic disorder characterized by impaired ciliary function, leading to chronic sinopulmonary disease. The genetic causes of PCD are still evolving, while the diagnosis is often dependent on finding a ciliary ultrastructural abnormality and immotile cilia. Here we report a novel gene associated with PCD but without ciliary ultrastructural abnormalities evident by transmission electron microscopy, but with dyskinetic cilia beating. METHODS: Genetic linkage analysis was performed in a family with a PCD subject. Gene expression was studied in Chlamydomonas reinhardtii and human airway epithelial cells, using RNA assays and immunostaining. The phenotypic effects of candidate gene mutations were determined in primary culture human tracheobronchial epithelial cells transduced with gene targeted shRNA sequences. Video-microscopy was used to evaluate cilia motion. RESULTS: A single novel mutation in CCDC65, which created a termination codon at position 293, was identified in a subject with typical clinical features of PCD. CCDC65, an orthologue of the Chlamydomonas nexin-dynein regulatory complex protein DRC2, was localized to the cilia of normal nasal epithelial cells but was absent in those from the proband. CCDC65 expression was up-regulated during ciliogenesis in cultured airway epithelial cells, as was DRC2 in C. reinhardtii following deflagellation. Nasal epithelial cells from the affected individual and CCDC65-specific shRNA transduced normal airway epithelial cells had stiff and dyskinetic cilia beating patterns compared to control cells. Moreover, Gas8, a nexin-dynein regulatory complex component previously identified to associate with CCDC65, was absent in airway cells from the PCD subject and CCDC65-silenced cells. CONCLUSION: Mutation in CCDC65, a nexin-dynein regulatory complex member, resulted in a frameshift mutation and PCD. The affected individual had altered cilia beating patterns, and no detectable ultrastructural defects of the ciliary axoneme, emphasizing the role of the nexin-dynein regulatory complex and the limitations of certain methods for PCD diagnosis