Comparative genomic analysis of Mycoplasma related to cell culture for infB gene-based loop-mediated isothermal amplification.

Mycoplasma contamination in cell culture affects the properties of cell lines. Gold standard detection by microbiological culture takes days and requires specialists. The polymerase chain reaction and loop-mediated isothermal amplification (LAMP) are fast molecular options, but LAMP only requires one heating block for DNA amplification. This study presents a comparative genomic analysis of Mycoplasma species to identify common target genes different from the rrsA gene, which encodes 16 S rRNA. The aim is to implement a LAMP assay to detect Mycoplasma species, reducing the time and specialized equipment required for detection. We performed a comparative genomic analysis through Mauve software and the GView server and selected infB and clpB genes as target candidates for designing LAMP primers. We evaluated both genes by multiple sequence alignment (MSA). The infB gene presented the best score MSA assessment with lower odd-log values (5,480,281) than other genes. We selected the infB gene to design LAMP primers specific to Mycoplasma spp. We used these primers to implement LAMP at 63 °C for 30 min, which showed 100% positive amplifications for detecting Mycoplasma spp. In conclusion, we present a methodology utilizing the infB gene-based LAMP assay to detect three of the six most prevalent Mycoplasma species in cell culture.

CRISPR-dCas9-Based Artificial Transcription Factors to Improve Efficacy of Cancer Treatment With Drug Repurposing: Proposal for Future Research.

Due to the high resistance that cancer has shown to conventional therapies, it is difficult to treat this disease, particularly in advanced stages. In recent decades, treatments have been improved, being more specific according to the characteristics of the tumor, becoming more effective, less toxic, and invasive. Cancer can be treated by the combination of surgery, radiation therapy, and/or drug administration, but therapies based on anticancer drugs are the main cancer treatment. Cancer drug development requires long-time preclinical and clinical studies and is not cost-effective. Drug repurposing is an alternative for cancer therapies development since it is faster, safer, easier, cheaper, and repurposed drugs do not have serious side effects. However, cancer is a complex, heterogeneous, and highly dynamic disease with multiple evolving molecular constituents. This tumor heterogeneity causes several resistance mechanisms in cancer therapies, mainly the target mutation. The CRISPR-dCas9-based artificial transcription factors (ATFs) could be used in cancer therapy due to their possibility to manipulate DNA to modify target genes, activate tumor suppressor genes, silence oncogenes, and tumor resistance mechanisms for targeted therapy. In addition, drug repurposing combined with the use of CRISPR-dCas9-based ATFs could be an alternative cancer treatment to reduce cancer mortality. The aim of this review is to describe the potential of the repurposed drugs combined with CRISPR-dCas9-based ATFs to improve the efficacy of cancer treatment, discussing the possible advantages and disadvantages.