End-to-end computational approach to the design of RNA biosensors for detecting miRNA biomarkers of cervical cancer.

Cervical cancer is a global public health subject as it affects women in the reproductive ages, and accounts for the second largest burden among cancer patients worldwide with an unforgiving 50% mortality rate. Relatively scant awareness and limited access to effective diagnosis have led to this enormous disease burden, calling for point-of-care, minimally invasive diagnosis methods. Here, an end-to-end quantitative unified pipeline for diagnosis has been developed, beginning with identification of optimal biomarkers, concurrent design of toehold switch sensors, and finally simulation of the designed diagnostic circuits to assess performance. Using miRNA expression data in the public domain, we identified miR-21-5p and miR-20a-5p as blood-based miRNA biomarkers specific to early-stage cervical cancer employing a multi-tier algorithmic screening. Synthetic riboregulators called toehold switches specific to the biomarker panel were then designed. To predict the dynamic range of toehold switches for use in genetic circuits as biosensors, we used a generic grammar of these switches, and built a neural network model of dynamic range using thermodynamic features derived from mRNA secondary structure and interaction. Second-generation toehold switches were used to overcome the design challenges associated with miRNA biomarkers. The resultant model yielded an adj. R2 ∼0.71, outperforming earlier models of toehold-switch dynamic range. Reaction kinetics modelling was performed to predict the sensitivity of the second-generation toehold switches to the miRNA biomarkers. Simulations showed a linear response between 10 nM and 100 nM before saturation. Our study demonstrates an end-to-end computational workflow for the efficient design of genetic circuits geared towards the effective detection of unique genomic/nucleic-acid signatures. The approach has the potential to replace iterative experimental trial and error, and focus time, money, and efforts. All software including the toehold grammar parser, neural network model and reaction kinetics simulation are available as open-source software (https://github.com/SASTRA-iGEM2019) under GNU GPLv3 licence.

Expression of CD44v6 but not E-cadherin or beta-catenin influences prognosis in primary pulmonary adenocarcinoma.

Primary pulmonary adenocarcinoma was studied, looking for relationships between the expression of cell adhesion molecules (CAMs) E-cadherin, beta-catenin and CD44v6, and clinicopathological tumour parameters and patient post-operative survival. Formalin-fixed, paraffin-embedded tissue from 120 primary lung adenocarcinomas, including 23 poorly differentiated tumours, 17 of probable bronchial origin, and 29 with a prominent bronchioloalveolar pattern, together with nodal metastatic tumour from 34 of these patients was stained using monoclonal antibodies and immunohistochemistry. Sections were scored either high level (>10% cells positive) or low level (<10% positive). High level expression of CD44v6 was retained in 28.4% (34/120) of tumours, while high levels of E-cadherin (57.5%, 69/120) and beta-catenin (80. 8%, 97/120) were more frequent. For all CAMs, staining levels did not correlate with nodal status, stage or tumour type. The apical or basal staining seen in normal bronchial and alveolar epithelium was often seen in papillary, glandular, and bronchioloalveolar areas of tumour, while solid invasive tumour more often showed pericellular staining. When the staining for each CAM in 34 nodal metastases was compared with that in the corresponding primary tumour, a high degree of concordance was found, with no tendency for metastases to show less staining than the primary tumour. Expression of E-cadherin and beta-catenin in the primary tumour had no influence on post-operative survival, but patients whose tumours had low level CD44v6 expression had a poorer post-operative survival than those with high levels of CD44v6 (p=0.0014 for all patients, p=0.0012 for stage I patients only). In primary pulmonary adenocarcinoma, the levels of expression of E-cadherin, beta-catenin, and CD44v6 are not associated with lymph node metastases or tumour stage but the staining pattern is associated with tumour morphology. Low levels of CD44v6 expression predict a poor post-operative survival, independently of stage, while there is no such relationship with the expression of E-cadherin or beta-catenin.