Fluorescence-Linked Aptamer Assay for SARS-CoV-2 Spike-Protein: A Step-by-Step Performance Analysis in Clinical Samples.

The COVID-19 pandemic has been a main concern over the last two years and has become one of the most important crises in the history of human health. Today, there is still a need for affordable and reliable diagnostic tests for massive disease monitoring. Previously, a set of highly specific DNA-aptamers (C7/C9) binding to the SARS-CoV-2 Spike (S) protein were isolated but its performance in clinical samples remained to be tested. Here, 242 samples were collected through three different methods and subjected to florescence-linked aptamer assays (FLAA) based on C7/C9 aptamers through two readout protocols. Then, a step-by-step statistical approach which included agreement tests, proportion comparisons and binomial and multinomial logistic regressions was used to predict optimal conditions for the novel C7/C9 FLAA test. RTqPCR threshold cycles, symptoms onset and processing time were influential factors on FLAA test results. Naturally occurring mutations on S were also detected and analyzed. Aminoacidic substitutions D614G and T732A appeared relevant for aptamer recognition although further studies are necessary. The methodology presented here is the first step to determine the performance and diagnosis across a range of clinical contexts and it might serve as a base for a complete analysis applicable to other designs of new diagnostic tests.

Simplified modeling of E. coli mortality after genome damage induced by UV-C light exposure.

UV light is a group of high-energy waves from the electromagnetic spectrum. There are three types of UV radiations: UV-A, -B and -C. UV-C light are the highest in energy, but most are retained by the ozone layer. UV-A and -B reach the earth's surface and cause damage on living organisms, being considered as mutagenic physical agents. Numerous test models are used to study UV mutagenicity; some include special lamps, cell cultures and mathematical modeling. Mercury lamps are affordable and useful sources of UV-C light due to their emission at near the maximum absorption peak of nucleic acids. E. coli cultures are widely used because they have DNA-damage and -repairing mechanisms fairly similar to humans. In here we present two simple models that describe UV-C light incidence on a genome matrix, using fundamental quantum-mechanical concepts and considering light as a particle with a discontinuous distribution. To test the accuracy of our equations, stationary phase cultures of several E. coli strains were exposed to UV-C light in 30 s-intervals. Surviving CFUs were counted and survival/mortality curves were constructed. These graphs adjusted with high goodness of fit to the regression predictions. Results were also analyzed using three main parameters: quantum yield, specific speed and time of mortality.

Aptamers coupled to nanoparticles in the diagnosis and treatment of microbial infections. / Aptámeros acoplados a nanopartículas para el diagnóstico y tratamiento de las infecciones microbianas.

There are nanoparticles with remarkable antibacterial characteristics and aptamers able to recognize specific pathogenic bacteria with high affinity and specificity. The combination of both systems has been used to design rapid bacterial detection methods with excellent detection limits. Likewise, the synergism between aptamers and nanoparticles have allowed to optimize the antimicrobial activity of antibiotics and other nanostructures providing them with activity bacterium-specific, turning into attractive and promising tools to fight against bacteria resistant to multiple antimicrobials.

Antimutagenic activity of vitamin B1 against damages induced by chemical and physical mutagens in Salmonella typhimurium and Escherichia coli.

Thiamine (vitamin B1) is an essential nutrient acting mainly as an enzymatic cofactor on diverse cell processes. It has been reported that vitamin B1 has a significant role in the signaling pathways related to the response to adverse environmental conditions (chemical and physical). The objectives of this study were to evaluate the antimutagenic potential of vitamin B1 in front of DNA-alkylating agents in the presence/absence of ogt and ada repairing genes in Salmonella typhimurium strains and against damage induced by ultraviolet light type C in Escherichia coli strains mutated at the uvrABC system and recBCD enzymes. For S. typhimurium, an antimutagenesis test (Ames test) was performed using strains deficient in one or both genes (YG7100 ada-/ogt+, YG7104 ada+/ogt-, YG7108 ada-/ogt-). For E. coli, mutated strains (K-12 derived strains Hfr H180 uvrB+/recA+, W3110 uvrB+/recA- and ATCC®8739 uvrB-/recA+) were exposed to UV-C light at different time intervals, with and without vitamin B1. Our results showed that thiamine is an antimutagen against methyl-N-nitro-N-nitrosoguanidine or ethyl-N-nitro-N-nitrosoguanidine only when the ogt gene is present. While for E. coli, the presence of vitamin B1 increased the survival rate, implying an antimutagenesis independent of uvrABC repairing system and recBCD enzymes.

Targeting of the HPV-16 E7 protein by RNA aptamers.

The expression of high-risk human papillomavirus E6 and E7 proteins in most cervical tumors raised a considerable interest in the diagnostic and therapeutic applications of functional oligonucleotides (i.e., DNAzymes, ribozymes, and aptamers) directed against HPV targets. Aptamers are short single-stranded oligonucleotides that specifically recognize a wide variety of molecular targets, including HPV proteins. Here, we describe a protocol for the successful isolation of RNA aptamers directed at the recombinant HPV-16 E7 protein through the application of the SELEX method. Once the nucleic acid sequence of a functional aptamer is determined, large amounts of the oligonucleotide can be produced and modified at low cost and high efficiency. The remarkable affinity and specificity of aptamers for their targets make these molecules the next-generation tool for diagnostics and therapeutics of cervical cancer.

Characterization of an RNA aptamer against HPV-16 L1 virus-like particles.

The human papillomavirus (HPV) capsid is mainly composed of the L1 protein that can self-assemble into virus-like particles (VLPs) that are structurally and immunologically similar to the infectious virions. We report here the characterization of RNA aptamers that recognize baculovirus-produced HPV-16 L1 VLPs. Interaction and slot-blot binding assays showed that all isolated aptamers efficiently bound HPV-16 VLPs, although the Sc5-c3 aptamer showed the highest specificity and affinity (Kd=0.05 pM). Sc5-c3 secondary structure consisted of a hairpin with a symmetric bubble and an unstructured 3'end. Biochemical and genetic analyses showed that the Sc5-c3 main loop is directly involved on VLPs binding. In particular, binding specificity appeared mediated by five non-consecutive nucleotide positions. Experiments using bacterial-produced HPV-16 L1 resulted in low Sc5-c3 binding, suggesting that recognition of HPV-16 L1 VLPs relies on quaternary structure features not present in bacteria-produced L1 protein. Sc5-c3 produced specific and stable binding to HPV-16 L1 VLPs even in biofluid protein mixes and thus it may provide a potential diagnostic tool for active HPV infection.

[Effect of cryotherapy over the expression of vascular endothelial growth factor and pigment epithelium-derived factor]. / Efecto de la crioterapia en la regulación de la expresión del factor de crecimiento del endotelio vascular y del factor derivado del epitelio pigmentado.

BACKGROUND: Cryotherapy is a no invasive technique that uses intense cold to freeze and destroy cancer tissues. There are no descriptions of its effects over the expression of vascular endothelial growth factor and pigment epithelium-derived factor. METHODS: Experimental study in cryogenic spot were applied in the right sclera of twelve pigs for ten minutes. Other 3 pigs were used as normal controls. Animals were sacrificed at 7, 14 and 21 and the tissues of choriodes and retina were dissected in areas of approximately 1 cm2 surrounding cryogenic spots. Expression levels of vascular endothelial growth factor and pigment epithelium-derived factor were determined analyzed using polymerase chain reaction coupled to reverse-transcription. RESULTS: Vascular endothelial growth factor was significantly downregulated (24%, p< 0.05) seven days post-treatment meanwhile pigment epithelium-derived factor levels increased 44.8% (p< 0.05) as compared to normal controls (untreated). Both vascular endothelial growth factor and pigment epithelium-derived factor levels remain the same until day 14 but returned to basal expression at day 21. DISCUSSION: This work expose the relation of cryotherapy with the expression of two factors related to angiogenesis. RESULTS showed significant changes on the expression of vascular endothelial growth factor and pigment epithelium-derived factor illustrating that both proteins are regulated in response to cryogenic treatment in relatively short periods (21 days).

Antecedentes: la crioterapia es una técnica no invasiva que usa frio intenso para congelar y destruir los tejidos cancerosos. Sus efectos en la expresión del factor de crecimiento del endotelio vascular y el factor derivado del epitelio pigmentado no se han descrito. Material y métodos: estudio experimental en modelos experimentales de crioterapia. En la esclera del ojo derecho de 12 cerdos se aplicó un punto de congelamiento durante 10 segundos. Se usaron 3 cerdos como controles normales. Los animales se sacrificaron a los 7, 14 y 21 días y el tejido de coroides y retina se seccionó en áreas de aproximadamente 1 cm2 circundantes al punto de congelamiento. Los niveles de expresión del factor de crecimiento del endotelio vascular y factor derivado del epitelio pigmentado se determinaron y analizaron por reacción en cadena de la polimerasa acoplada a reverso-transcripción. Resultados: los niveles de factor de crecimiento del endotelio vascular disminuyeron significativamente (24%, p < 0.05) a los 7 días postratamiento, mientras que la expresión del factor derivado del epitelio pigmentado aumentó 44.8% (p< 0.05) en comparación con los niveles de las muestras normales. Los niveles de expresión se mantuvieron hasta el día 14 y regresaron a valores basales en el día 21. Conclusiones: este trabajo expone la relación entre la crioterapia y la expresión de dos factores angiogénicos. Los resultados muestran cambios significativos en la expresión del factor de crecimiento del endotelio vascular y factor derivado del epitelio pigmentado, y evidencian que ambas proteínas son reguladas en respuesta al tratamiento criogénico en periodos relativamente cortos (21 días).