Genome-wide CRISPR screens identify the YAP/TEAD axis as a driver of persister cells in EGFR mutant lung cancer.

Most lung cancer patients with metastatic cancer eventually relapse with drug-resistant disease following treatment and EGFR mutant lung cancer is no exception. Genome-wide CRISPR screens, to either knock out or overexpress all protein-coding genes in cancer cell lines, revealed the landscape of pathways that cause resistance to the EGFR inhibitors osimertinib or gefitinib in EGFR mutant lung cancer. Among the most recurrent resistance genes were those that regulate the Hippo pathway. Following osimertinib treatment a subpopulation of cancer cells are able to survive and over time develop stable resistance. These 'persister' cells can exploit non-genetic (transcriptional) programs that enable cancer cells to survive drug treatment. Using genetic and pharmacologic tools we identified Hippo signalling as an important non-genetic mechanism of cell survival following osimertinib treatment. Further, we show that combinatorial targeting of the Hippo pathway and EGFR is highly effective in EGFR mutant lung cancer cells and patient-derived organoids, suggesting a new therapeutic strategy for EGFR mutant lung cancer patients.

Identifying Behavioural Traits and Underlying Personality Dimensions in Domestic Ferrets (Mustela putorius furo).

The aim of this study was to examine the personality structure of domestic ferrets (Mustela putorius furo) by using owner-based reporting of personality traits. A total of 743 ferret owners participated in an online questionnaire, with a total of 1029 ferrets being assessed. Respondents rated 67 adjectives based on their ferret(s) behavioural traits and personality. Principal component analysis (PCA) of these trait ratings identified four underlying personality dimensions, which accounted for 47.1% of the total variance. These were labelled according to the traits that they encompass: Extraversion, Sociability, Attentiveness, and Neuroticism. Details about ferret sex, de-sexing status, age, and coat colour were also sought, and General Linear Mixed Models were used to test the main effects of these characteristics on the personality dimensions. It was found that sex (p < 0.01) and age (p < 0.001) significantly influenced certain personality components, whereas de-sexing did not. Sociability, Attentiveness, and Neuroticism were found to differ based on sex, whereby males were rated as more sociable than females, but females were rated higher on the Attentiveness and Neuroticism subscales. Finally, Extraversion was found to generally decrease with age. We can use the findings of this study to make cross-species comparisons and further inform the discussion regarding the adaptive relevance of animal personality. Identifying differences in personality types can improve welfare by using this information to satisfy the different needs of individuals.

FluChip-8G Insight: HA and NA subtyping of potentially pandemic influenza A viruses in a single assay.

BACKGROUND: Global influenza surveillance in humans and animals is a critical component of pandemic preparedness. The FluChip-8G Insight assay was developed to subtype both seasonal and potentially pandemic influenza viruses in a single assay with a same day result. FluChip-8G Insight uses whole gene segment RT-PCR-based amplification to provide robustness against genetic drift and subsequent microarray detection with artificial neural network-based data interpretation. OBJECTIVES: The objective of this study was to verify and validate the performance of the FluChip-8G Insight assay for the detection and positive identification of human and animal origin non-seasonal influenza A specimens. METHODS: We evaluated the ability of the FluChip-8G Insight technology to type and HA and NA subtype a sample set consisting of 297 results from 180 unique non-seasonal influenza A strains (49 unique subtypes). RESULTS: FluChip-8G Insight demonstrated a positive percent agreement ≥93% for 5 targeted HA and 5 targeted NA subtypes except for H9 (88%), and negative percent agreement exceeding 95% for all targeted subtypes. CONCLUSIONS: The FluChip-8G Insight neural network-based algorithm used for virus identification performed well over a data set of 297 naïve sample results, and can be easily updated to improve performance on emerging strains without changing the underlying assay chemistry.

Clinical validation of the FluChip-8G Influenza A+B Assay for influenza type and subtype identification.

BACKGROUND: The FluChip-8G Influenza A+B Assay is a multiplexed influenza RT-PCR and microarray-based assay with same day turnaround time, developed to subtype seasonal A viruses (H1N1pdm2009 and H3N2), distinguish B viruses as Yamagata or Victoria lineage, and is the only FDA cleared assay capable of positive identification of a wide variety of A subtypes as "non-seasonal" A viruses from human nasal specimens. OBJECTIVE: To evaluate clinical performance of the FluChip-8G Influenza A+B Assay for detection of seasonal influenza viruses in nasal and nasopharyngeal swab specimens, and to evaluate performance for detection of non-seasonal influenza viruses using contrived samples. STUDY DESIGN: For seasonal viruses, a multisite study of the FluChip-8G Influenza A+B Assay using prospectively and retrospectively collected nasal and nasopharyngeal swabs was performed using the FDA-cleared CDC Human Flu Dx Panel as the comparator assay. For non-seasonal viruses, testing was performed at a single site using contrived samples from 100 unique non-seasonal strains representing 41 subtypes. RESULTS: Sensitivity (95% CI) and specificity (95% CI) for each target group, respectively, from results of 1689 clinical specimens were: seasonal H1N1pdm2009: 96.4% (87.9-99.0), 99.3% (98.8-99.6), seasonal H3N2: 91.8% (87.7-94.7), 99.7% (99.2-99.9), Influenza B Victoria: 100% (94.0-100.0), 99.9% (99.6-100.0), and Influenza B Yamagata: 95.6% (89.2-98.3), 99.9% (99.6-100.0). The sensitivity and specificity from contrived influenza A non-seasonal viruses was determined to be 99.0% (94.6-99.8) and 100% (96.7-100.0). CONCLUSION: The FluChip-8G Influenza A+B Assay has robust sensitivity and specificity for detecting and identifying all target virus groups, including non-seasonal influenza A, with same day results.

Analytical evaluation of the microarray-based FluChip-8G Influenza A+B Assay.

BACKGROUND: Influenza causes a significant annual disease burden, with characterization of the infecting virus important in clinical and public health settings. Rapid immunoassays are fast but insensitive, whereas real-time RT-PCR is sensitive but susceptible to genetic mutations and often requires multiple serial assays. The FluChip-8G Influenza A+B Assay provides type and subtype/lineage identification of influenza A and B, including non-seasonal A viruses, in a single microarray-based assay with same day turnaround time. OBJECTIVE: To evaluate key analytical performance characteristics of the FluChip-8G Influenza A+B Assay. STUDY DESIGN: Analytical sensitivity, cross-reactivity, and multi-site reproducibility were evaluated. RESULTS: The limit of detection (LOD) for the FluChip-8G influenza A+B Assay ranged from 5.8 × 102-1.5 × 105 genome copies/mL, with most samples ∼2 × 103 genome copies/mL (∼160 genome copies/reaction). Fifty two (52) additional strains were correctly identified near the LOD, demonstrating robust reactivity. Two variant viruses (H1N1v and H3N2v) resulted in dual identification as both "non-seasonal influenza A" and A/H1N1pdm09. No reproducible cross-reactivity was observed for the 34 organisms tested, however, challenges with internal control inhibition due to crude growth matrix were observed. Lastly, samples tested near the LOD showed high reproducibility (97.0% (95% CI 94.7-98.7)) regardless of operator, site, reagent lot, or testing day. CONCLUSION: The FluChip-8G Influenza A+B Assay is an effective new method for detecting and identifying both seasonal and non-seasonal influenza viruses, as revealed by good sensitivity and robust reactivity to 52 unique strains of influenza virus. In addition, the lack of cross-reactivity to non-influenza pathogens and high lab-to-lab reproducibility highlight the analytical performance of the assay as an alternative to real-time RT-PCR and sequencing-based assays. Clinical validation of the technology in a multi-site clinical study is the subject of a separate investigation.

Impact of trivalent arsenicals on selenoprotein synthesis.

BACKGROUND: Exposure to arsenic has been associated with development of skin, lung, bladder, liver, and kidney cancer. Recent evidence suggests that an increase in oxidative stress in cells treated with arsenicals represents the molecular mechanism behind arsenic-induced carcinogenesis. Selenium, in the form of selenocysteine, is necessary for the activity of several enzymes with a role in defense against reactive oxygen species. A mutual sparing effect between arsenic and selenium has been shown in animal studies when both metalloids are present in high concentrations. OBJECTIVES: To determine whether changes in selenoprotein synthesis may be an underlying mechanism behind arsenic-induced carcinogenesis, we analyzed the new synthesis of selenoproteins within cells after exposure to inorganic or methylated arsenicals using a human keratinocyte cell model. RESULTS: Addition of arsenite to culture medium blocked new synthesis of selenoproteins when selenium was present in the form of selenite, and appeared to stimulate the use of serum-derived selenium. Monomethylarsonous acid (MMA(III)) treatment of cells, in contrast, did not block all new synthesis of selenoproteins but did result in an increase in cytosolic thioredoxin reductase (TrxR1) at both the mRNA and protein levels. MMA(III) also reduced the new synthesis of cellular glutatione peroxidase (cGpx) and other smaller selenoproteins. Dimethylarsinous acid (DMA(III)) stimulated selenoprotein synthesis by an as yet unknown mechanism. CONCLUSIONS: These results suggest that arsenite and MMA(III) are key metabolites that trigger higher levels of TrxR1, and both lead to a reduction in the expression of cGpx. Together these effects certainly could lead to carcinogenesis given the knowledge that many cancers have higher levels of TrxR, and reduced Gpx levels will reduce the cell's ability to defend against reactive oxygen species. Based on these results, the impact of the trivalent arsenicals arsenite and MMA(III) on selenoprotein synthesis may indeed represent a potential molecular mechanism for the higher rates of cancer observed in populations exposed to high levels of arsenic.