The Automatic Tracking Of <i>Caenorhabditis elegans</i> And Its Use In Determining Genetic Function

Even with its simple nervous system, the nematode worm Caenorhabditis elegans can display a range of complex behaviours. Movement can be viewed as the main output of the C. elegans nervous system, and aberrations in the worm’s locomotion can be used as an indicator for genetic function in mutant strains of C. elegans. Automated tracking of C. elegans locomotion has been used to determine phenotypic fingerprints for ~300 mutant C. elegans strains. Two methods of creating phenotypic fingerprints were used. The first based on pre-determined micro-behaviours previously described in worms, but never before analysed using automated tracking. The second used the tracking data itself to determine micro-motifs, repeated sets of behaviours observed at least twice in at least two mutant or wild-type strains. Both methods of clustering successfully grouped together strains with mutations in genes known to interact together, verifying that the technique is able to detect meaningful connections between mutant strains. The following step was to determine whether the technique can be used to establish connections between genes on unknown function. A pair of strains with mutations in DEG/ENaC subunit encoding genes clustered strongly together using the micro-motif method, due to similar defects in their behaviours upon turning. The function of these genes, asic-2 and acd-5, was unknown. Upon further investigation it was found that the two genes are expressed in different classes of neurons, the IL2s in the case of asic-2 and the ASIs in the case of acd-5. Following investigation into behaviours known to be modulated by these two neuron classes it was found that the mutant strains displayed mutant phenotypes in similar behaviours, but that their mutant phenotypes are opposing. Mutations in asic-2 cause increased lifespan and healthspan and a reduction in dauer entry in response to exogenous, purified ascarosides. Mutations in acd-5 cause decreased lifespan and healthspan and a reduction in dauer entry in response to crude dauer pheromone. This suggested that the two genes were unlikely to be working in the same pathway, but do function in similar pathways. Calcium imaging is a technique used in C. elegans to measure responses in excitable cells, in this case in neurons. Many calcium indicators are available for use in this technique, one in particular is GCaMP. GCaMP has undergone many rounds of targeted mutations with the aim to increase the molecule’s dynamic range and dissociation constant. At the time of commencing this project, new variants of GCaMP, known as GCaMP6s, became available, and had yet to be tested in C. elegans neurons. The effectiveness of a total of 6 new variants was tested in the gentle touch neurons of C. elegans. It was found that the alterations made to GCaMP5G in order to make the GCaMP6 variants did not result in improved dynamic range or dissociation constant in the PLM of C. elegans

Serum proteomics and plasma fibulin-3 in differentiation of mesothelioma from asbestos-exposed controls and patients with other pleural diseases

Introduction: Malignant pleural mesothelioma (MPM) is difficult to diagnose. An accurate blood biomarker could prompt specialist referral or be deployed in future screening. In earlier retrospective studies, SOMAscan proteomics (Somalogic, Boulder, CO) and fibulin-3 seemed highly accurate, but SOMAscan has not been validated prospectively and subsequent fibulin-3 data have been contradictory. Methods: A multicenter prospective observational study was performed in 22 centers, generating a large intention-to-diagnose cohort. Blood sampling, processing, and diagnostic assessment were standardized, including a 1-year follow-up. Plasma fibulin-3 was measured using two enzyme-linked immunosorbent assays (CloudClone [used in previous studies] and BosterBio, Pleasanton, CA). Serum proteomics was measured using the SOMAscan assay. Diagnostic performance (sensitivity at 95% specificity, area under the curve [AUC]) was benchmarked against serum mesothelin (Mesomark, Fujirebio Diagnostics, Malvern, PA). Biomarkers were correlated against primary tumor volume, inflammatory markers, and asbestos exposure. Results: A total of 638 patients with suspected pleural malignancy (SPM) and 110 asbestos-exposed controls (AECs) were recruited. SOMAscan reliably differentiated MPM from AECs (75% sensitivity, 88.2% specificity, validation cohort AUC 0.855) but was not useful in patients with differentiating non-MPM SPM. Fibulin-3 (by BosterBio after failed CloudClone validation) revealed 7.4% and 11.9% sensitivity at 95% specificity in MPM versus non-MPM SPM and AECs, respectively (associated AUCs 0.611 [0.557–0.664], p = 0.0015) and 0.516 [0.443–0.589], p = 0.671), both inferior to mesothelin. SOMAscan proteins correlated with inflammatory markers but not with asbestos exposure. Neither biomarker correlated with tumor volume. Conclusions: SOMAscan may prove useful as a future screening test for MPM in asbestos-exposed persons. Neither fibulin-3 nor SOMAscan should be used for diagnosis or pathway stratification