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Additional file 1: Fig. S1. Amplication of the cofD gene of M. ruminantium by using cofD primers. Fig. S2. The recombinant plasmid pEASY-T1-cofD was amplified using cofD-F and cofD-R as primers, and the transformants were identified by PCR. Fig. S3. PCR product sequencing of positive clones and comparison of similarities of cofD by Blast. Fig. S4. Identification of 9 monoclonal recombinant plasmids pUCl8-cofD-tet selected by PCR using tet specific primers. Fig. S5. Double digestion of pUCl8-cofD-tet recombinant plasmid DNA using EcoRI and HindIII, detection by 1% agarose gel electrophoresis. (DOCX 437 KB)

Additional file 2 Table S2. The primers used in the construction of Illumina sequencing library.

The nucleic acid guanine-quadruplex structures (G4s) are involved in many aspects of cancer progression. The DEAH-box polypeptide 36 (DHX36) has been identified as a dominant nucleic acid helicase which targets and disrupts DNA and RNA G4s in an ATP-dependent manner. However, the actual role of DHX36 in breast cancer remains unknown. In this study, we observed that the gene expression of DHX36 was positively associated with patient survival in breast cancer. The abundance of DHX36 is also linked with pathologic conditions and the stage of breast cancer. By using the xenograft mouse model, we demonstrated that the stable knockdown of DHX36 via lentivirus in breast cancer cells significantly promoted tumour growth. We also found that, after the DHX36 knockdown (KD), the invasion of triple-negative breast cancer cells was enhanced. In addition, we found a significant increase in the number of cells in the S-phase and a reduction of apoptosis with the response to cisplatin. DHX36 KD also desensitized the cytotoxic cellular response to paclitaxel and cisplatin. Transcriptomic profiling analysis by RNA sequencing indicated that DHX36 altered gene expression profile through the upstream activation of TNF, IFNγ, NFκb and TGFβ1. High throughput signalling analysis showed that one cluster of stress-associated kinase proteins including p53, ROCK1 and JNK were suppressed, while the mitotic checkpoint protein-serine kinases CDK1 and CDK2 were activated, as a consequence of the DHX36 knockdown. Our study reveals that DHX36 functions as a tumour suppressor and may be considered as a potential therapeutic target in breast cancer.

Lan-Lan Zhong,1,2,* Qian Zhou,3,*Cui-Yan Tan,3,*Adam P Roberts,4,5Mohamed Abd El-Gawad El-Sayed Ahmed,1–2,6 Guanping Chen,1,2Min Dai,7 Fan Yang,8 Yong Xia,9 Kang Liao,10 Yingjian Liang,3 Yongqiang Yang,1,11Siyuan Feng,1,2Xiaobin Zheng,3,*Guo-Bao Tian1,2,*1Program in Pathobiology, The Fifth Affiliated Hospital, Zhongshan School of Medicine, Sun Yat-Sen University, Guangdong 519000, People’s Republic of China; 2Ministry of Education, Key Laboratory of Tropical Diseases Control, Sun Yat-sen University, Guangzhou 510080, People’s Republic of China; 3Department of Respiratory Medicine, The Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai 519000, People’s Republic of China; 4Department of Tropical Disease Biology, Liverpool School of Tropical Medicine, Liverpool, L3 5QA, UK; 5Centre for Drugs and Diagnostics, Liverpool School of Tropical Medicine, Liverpool L3 5QA, UK; 6Department of Microbiology and Immunology, Faculty of Pharmaceutical Sciences and Drug Manufacturing, Misr University for Science and Technology (MUST), Cairo, Egypt; 7School of Laboratory Medicine, Chengdu Medical College, Chengdu 610500, People’s Republic of China; 8Department of Microbiology, School of Basic Medical Science, Xinxiang Medical University, Xinxiang 453003, People’s Republic of China; 9Department of Clinical Laboratory Medicine, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou 510150, People’s Republic of China; 10Department of Clinical Laboratory, The First Affiliated Hospital of Sun Yat-Sen University, Guangzhou 510080, People’s Republic of China; 11School of Pharmaceutical Sciences (Shenzhen), Sun Yat-sen University, Guangzhou 510006, People’s Republic of China*These authors contributed equally to this workPurpose: The discovery of the plasmid-mediated colistin resistance genes, mcr, revealed a mechanism of transmission of colistin resistance, which is a major, global public health concern especially among individuals infected with carbapenem-resistant Gram-negative bacteria. To monitor the spread and epidemiology of mcr genes, a convenient and reliable method to detect mcr genes in clinical isolates is needed, especially in the primary care institutions. This study aimed to establish a restriction endonuclease-based multiplex loop-mediated isothermal amplification (multi-LAMP) assay to detect mcr genes (mcr-1 to mcr-5) harbored by colistin-resistant bacteria.Methods: A triple-LAMP assay for mcr-1, mcr-3, and mcr-4 and a double-LAMP assay for mcr-2 and mcr-5 were established. The sensitivity and specificity of the LAMP reactions were determined via electrophoresis and visual detection.Results: The sensitivity of the LAMP assay was 10-fold greater than that of PCR, with high specificity among the screened primers. Specific mcr genes were distinguished in accordance with band numbers and the fragment length of the digested LAMP amplification products. Furthermore, the LAMP assay was confirmed as a rapid and reliable diagnostic technique upon application for clinical samples, and the results were consistent with those of conventional PCR assay.Conclusion: The multi-LAMP assay is a potentially promising method to detect mcr genes and will, if implemented, help prevent infections by drug-resistant bacteria in primary-care hospitals due to rapid and reliable surveillance. To our knowledge, this is the first study to report the application of LAMP to detect mcr-2 to mcr-5 genes and the first time that multi-LAMP has been applied to detect mcr genes.Keywords: mcr genes, colistin resistance, multi-LAMP, rapid detection, enzyme digestion

Figure S1. Point assignment and prognostic score for DFS and OS. (DOCX 15 kb)