Commercially available industry-relevant software in the education of genome variant curation

BACKGROUND Genome analytics is a drastically expanding field, and there is an increasing demand for individuals with the necessary skillset to analyse the genome data that is being generated. A new Masters by coursework was developed to train individuals in genome analytics. Institutions analysing genomes commonly utilise in-house analysis tools, but increasingly commercial software packages that integrate AI are being considered in the research and diagnostic space (De La Vega et al., 2021). AIMS Evaluate the effectiveness of commercially available software as a tool for teaching variant analysis, classification and curation, enabling the analysis of real-world case examples for the teaching, training and assessment of students in the field of diagnostic genome analysis.   Assess students’ perspectives on work readiness after using commercially available tools in the educational environment. DESIGN AND METHODS Students were exposed to the use and limitations of a commercial software package for Human genome curation during two core units of the course. This software was used as part of both in-class training and in their assessment case studies. Students were invited to voluntarily complete an online survey including qualitative and quantitative components featuring Likert scale questions, both pre and post exposure to the software. Paired data from 23 individuals (73% response rate), most aged between 18 and 25, were recorded and anonymised prior to analysis. Qualitative data were thematically coded blind by two individuals independently using emergent coding (Charmaz, 2008). RESULTS AND CONCLUSIONS This project indicates that after the completion of the units that integrated commercially available industry software, we measured increased student confidence (increase in percentage reporting fairly confident or higher) in joining the genetic analysis workforce (significant change from 37% to 70%) and in completing job-specific tasks (significant increase in 7 out of 9 tasks of between 28% to 39%). The aspects of their studies the students valued in relation to these changes and their perception of the usefulness of integration of the commercial software were elucidated from qualitative theming, and can inform others looking to integrate commercially available software within their tertiary degree. REFERENCES Charmaz, K. (2008).  Grounded theory as an emergent method. In S. N. Hesse-Biber & P. Leavy (Eds.), Handbook of emergent methods. (pp. 155-170). The Guilford Press.  De La Vega, F.M., Chowdhury, S., Moore, B., Frise, E., McCarthy, J., Hernandez, E.J., Wong, T., James, K., Guidugli, L., Agrawal, P.B., Genetti, C.A., Brownstein, C.A., Beggs, A.H., Löscher, B.S., Franke, A., Boone, B., Levy, S.E., Õunap, K., Pajusalu, S., … Kingsmore, S.F. (2021). Artificial intelligence enables comprehensive genome interpretation and nomination of candidate diagnoses for rare genetic diseases. Genome Med, 13(1), 153. https://doi.org/10.1186/s13073-021-00965-

Discriminating the earliest stages of mammary carcinoma using myoepithelial and proliferative markers.

Mammographic screening has led to increased detection of breast cancer at a pre-invasive state, hence modelling the earliest stages of breast cancer invasion is important in defining candidate biomarkers to predict risk of relapse. Discrimination of pre-invasive from invasive lesions is critically important for such studies. Myoepithelial cells are the barrier between epithelial cells and the surrounding stroma in the breast ductal system. A number of myoepithelial immunohistochemistry markers have been identified and validated in human tissue for use by pathologists as diagnostic tools to distinguish in situ carcinoma from invasive breast cancer. However, robust myoepithelial markers for mouse mammary tissue have been largely under-utilised. Here, we investigated the utility of the myoepithelial markers smooth muscle actin (SMA), smooth muscle myosin heavy chain (SMMHC), cytokeratin-14 (CK14) and p63 to discriminate mammary intraepithelial neoplasia (MIN) from invasive disease in the C57BL/6J MMTV-PyMT transgenic model of mammary carcinoma. We identified that SMMHC and CK14 are retained in early in situ neoplasia and are appropriate markers for distinguishing MIN from invasive disease in this model. Additionally, the proliferation marker Ki67 is a superior marker for differentiating between normal and hyperplastic ducts, prior to the development of MIN. Based on this, we developed a scoring matrix for discriminating normal, hyperplasia, MIN and invasive lesions in this spontaneous mammary tumorigenesis model. This study demonstrates heterogeneous expression of myoepithelial proteins throughout tumour development, and highlights the need to characterise the most appropriate markers in other models of early breast cancer to allow accurate classification of disease state

Myoepithelial markers on mouse mammary gland tissue.

<p>Sections of formalin-fixed, paraffin-embedded Balb/c and C57BL/6J mammary gland tissue were stained with a panel of myoepithelial markers, smooth muscle actin (SMA), smooth muscle myosin heavy chain (SMMHC) and cytokeratin 14 (CK14) and visualized with DAB (brown staining). All sections were counterstained with hematoxylin. Scale bar represents 50 μm.</p

Scoring matrix to distinguish different tumour stages in C57BL/6J MMTV-PyMT mice.

<p>Scoring matrix to distinguish different tumour stages in C57BL/6J MMTV-PyMT mice.</p

Expression of myoepithelial markers and Ki67 in C57BL/6J MMTV-PyMT mice.

<p>Expression of myoepithelial markers and Ki67 in C57BL/6J MMTV-PyMT mice.</p

Identification of mouse myoepithelial markers in C57BL/6J MMTV-PyMT mammary gland tissue.

<p>Sections of formalin-fixed, paraffin-embedded C57BL/6J MMTV-PyMT mammary gland tissue were stained with Ki67 and a panel of myoepithelial markers and visualized with DAB (brown staining). All sections were counterstained with hematoxylin. Tissue sections containing normal ducts, hyperplastic, MIN and invasive regions (determined by a pathologist) were compared to determine the clearest myoepithelial markers for mouse tissue. Scale bar represents 50 μm.</p

Scoring SMMHC and CK14 expression to distinguish MIN from invasive lesions in C57BL/6J MMTV-PyMT mice.

<p>The expression of SMMHC and CK14 in myoepithelial cells were scored and averaged from independent areas from five mice. Two-sided Fisher’s exact test, p = 0.0031 (**p<0.01) <i>n</i> = 5 mice.</p

Bifluoride Ion Mediated SuFEx Trifluoromethylation of Sulfonyl Fluorides and Iminosulfur Oxydifluorides

Sulfur-Fluoride Exchange (SuFEx) is the new generation click chemistry transformation exploiting the unique properties of S-F bonds and their ability to undergo near-perfect reactions with nucleophiles. We report here the first SuFEx based protocol for the efficient synthesis of pharmaceutically important triflones and bis(trifluoromethyl)sulfur oxyimines from the corresponding sulfonyl fluorides and iminosulfur oxydifluorides, respectively. The new protocol involves the rapid exchange of the S-F bond with trifluoromethyltrimethylsilane (TMSCF3) upon activation with potassium bifluoride in anhydrous DMSO. The reaction tolerates a wide selection of substrates and proceeds under mild conditions without need for chromatographic purification. A tentative catalytic mechanism is proposed supported by DFT calculations, involving formation of the free trifluoromethyl anion followed by nucleophilic displacement of the S-F through a five-coordinate intermediate. The preparation of a benzothiazole derived bis(trifluoromethyl)sulfur oxyimine with cytotoxic selectivity for MCF7 breast cancer cells demonstrates the utility of this methodology for the late-stage functionalization of bioactive molecules

Bifluoride Ion Mediated SuFEx Trifluoromethylation of Sulfonyl Fluorides and Iminosulfur Oxydifluorides

Sulfur-Fluoride Exchange (SuFEx) is the new generation click chemistry transformation exploiting the unique properties of S-F bonds and their ability to undergo near-perfect reactions with nucleophiles. We report here the first SuFEx based protocol for the efficient synthesis of pharmaceutically important triflones and bis(trifluoromethyl)sulfur oxyimines from the corresponding sulfonyl fluorides and iminosulfur oxydifluorides, respectively. The new protocol involves the rapid exchange of the S-F bond with trifluoromethyltrimethylsilane (TMSCF3) upon activation with potassium bifluoride in anhydrous DMSO. The reaction tolerates a wide selection of substrates and proceeds under mild conditions without need for chromatographic purification. A tentative catalytic mechanism is proposed supported by DFT calculations, involving formation of the free trifluoromethyl anion followed by nucleophilic displacement of the S-F through a five-coordinate intermediate. The preparation of a benzothiazole derived bis(trifluoromethyl)sulfur oxyimine with cytotoxic selectivity for MCF7 breast cancer cells demonstrates the utility of this methodology for the late-stage functionalization of bioactive molecules.<br /