The non-vesicle cell-free DNA (cfDNA) induces cell transformation associated with horizontal DNA transfer.

BACKGROUND: Cell-free DNA (cfDNA) is a source for liquid biopsy used for cancer diagnosis, therapy selection, and disease monitoring due to its non-invasive nature and ease of extraction. However, cfDNA also participates in cancer development and progression by horizontal transfer. In humans, cfDNA circulates complexed with extracellular vesicles (EV) and macromolecular complexes such as nucleosomes, lipids, and serum proteins. The present study aimed to demonstrate whether cfDNA not associated with EV induces cell transformation and tumorigenesis. METHODS: Supernatant of the SW480 human colon cancer cell line was processed by ultracentrifugation to obtain a soluble fraction (SF) and a fraction associated with EV (EVF). Primary murine embryonic fibroblast cells (NIH3T3) underwent passive transfection with these fractions, and cell proliferation, cell cycle, apoptosis, cell transformation, and tumorigenic assays were performed. Next, cfDNA was analyzed by electronic microscopy, and horizontal transfer was assessed by human mutant KRAS in recipient cells via PCR and recipient cell internalization via fluorescence microscopy. RESULTS: The results showed that the SF but not the EVF of cfDNA induced proliferative and antiapoptotic effects, cell transformation, and tumorigenesis in nude mice, which were reduced by digestion with DNAse I and proteinase K. These effects were associated with horizontal DNA transfer and cfDNA internalization into recipient cells. CONCLUSIONS: The results suggest pro-tumorigenic effects of cfDNA in the SF that can be offset by enzyme treatment. Further exploration of the horizontal tumor progression phenomenon mediated by cfDNA is needed to determine whether its manipulation may play a role in cancer therapy.

End-point RT-PCR based on a conservation landscape for SARS-COV-2 detection.

End-point RT-PCR is a suitable alternative diagnostic technique since it is cheaper than RT-qPCR tests and can be implemented on a massive scale in low- and middle-income countries. In this work, a bioinformatic approach to guide the design of PCR primers was developed, and an alternative diagnostic test based on end-point PCR was designed. End-point PCR primers were designed through conservation analysis based on kmer frequency in SARS-CoV-2 and human respiratory pathogen genomes. Highly conserved regions were identified for primer design, and the resulting PCR primers were used to amplify 871 nasopharyngeal human samples with a previous RT-qPCR based SARS-CoV-2 diagnosis. The diagnostic test showed high accuracy in identifying SARS-CoV-2-positive samples including B.1.1.7, P.1, B.1.427/B.1.429 and B.1.617.2/ AY samples with a detection limit of 7.2 viral copies/µL. In addition, this test could discern SARS-CoV-2 infection from other viral infections with COVID-19-like symptomatology. The designed end-point PCR diagnostic test to detect SARS-CoV-2 is a suitable alternative to RT-qPCR. Since the proposed bioinformatic approach can be easily applied in thousands of viral genomes and over highly divergent strains, it can be used as a PCR design tool as new SARS-CoV-2 variants emerge. Therefore, this end-point PCR test could be employed in epidemiological surveillance to detect new SARS-CoV-2 variants as they emerge and propagate.

Impact of the GK-1 adjuvant on peritoneal macrophages gene expression and phagocytosis.

PURPOSE: The synthetic peptide GK-1 potentiates protective immunity elicited by the influenza vaccine in mice. In order to understand its adjuvant properties, this study was designed to determine the impact of GK-1 on gene expression and phagocytosis of peritoneal macrophages (PMa). METHODS: Increased gene expression of chemokines involved in leukocyte recruitment and of pro-inflammatory mediators was detected by microarray analysis of control and GK-1 treated PMa macrophages. The expression profile was subsequently confirmed by Multiplex Immunoassays analysis to measure cytokines levels, flow cytometer to describe M1/M2 surface markers and an assay to evaluate their phagocytic activity. RESULTS: Treatment of PMa with GK-1 results in development to the classically activated M1 functional macrophage subpopulation with increased expression of the CCL3 and CXCLO2 chemokines, IL-6 and TNF-α proinflammatory cytokines with a concomitant increase in the levels of NO, accompanied by the expression of modulatory factors that downregulate the inflammatory phenotype. GK-1 treated PMa significantly increased their phagocytic activity. CONCLUSION: GK-1 classical activated with enhanced phagocitic capacity may underlie in the increased specific immunity induced when concomitant administered with other antigens.