Biology subjects

Mendez-Sanchez, S. C.

Publications and source records attributed to Mendez-Sanchez, S. C..

3 recordsLinked to original sources

bioRxiv2024

TropD-Detector: A CRISPR/LbCas12a-Based system for rapid and sensitive screening of Trypanosoma cruzi in Chagas vectors and reservoirs

Chagas disease, also known as American Trypanosomiasis, is a zoonosis with global distribution caused by the parasite Trypanosoma cruzi, primarily transmitted through the feces of infected triatomines. The emergence of new cases in non-endemic areas highlights the importance of early pathogen detection in vectors and reservoirs to generate effective control strategies and establish preventive policies. The objective of this study was to design and validate a detection system of T. cruzi based on specific DNA cleavage and collateral activation using the CRISPR/LbCas12a system, targeting the genes Cytochrome B (Cytb), 18S ribosomal subunit (SR18s), and histone (H2A). This system was validated for their uses in both vectors and reservoirs of the parasite. The first step was to conduct a bioinformatic analysis of the target genes and design RNA guides for each cleavage site and primers to amplify the target region through PCR. Subsequently, we sequenced the amplified DNA target and validated the detection system using T. cruzi DNA extracted from naturally infected Rhodnius pallescens in the metropolitan area of Bucaramanga, Colombia. After standardizing the method, we tested the CRISPR/Cas system with Silvio X10/4 laboratory strain of T. cruzi and scaled up to blood samples of naturally infected Didelphis marsupialis. As a result, we observed the DNA cleavage employing the CRISPR/Cas system with Cytb guide with a sensitivity of up to 0.0475 ng/uL of provided DNA. Sequencing of the Cytb gene showed no mutations in the cleavage site. However, point mutations and indels were found in SR18S and H2A, avoiding the formation of the CRISPR/LbCas12 complex. Furthermore, we introduce the design of a fluorescent detection prototype with CRISPR/LbCas12a called "Tropical Diseases Detector" (TropD-Detector). This device operates with an excitation wavelength of 480 nm emitted by an LED and a high-pass light filter with a cutoff wavelength of 500 nm. We detected positive samples using any photographic camera system. The TropD-Detector provides a visual, viable, and sensitive method for detecting T. cruzi in both vectors and reservoirs from endemic areas.

molecular biology↗

bioRxiv2020

Spatial control of gene expression in flies using bacterially derived binary transactivation systems.

Controlling gene expression is an instrumental tool for biotechnology, as it enables the dissection of gene function, affording precise spatial-temporal resolution. To generate this control, binary transactivational systems have been used employing a modular activator consisting of a DNA binding domain(s) fused to activation domain(s). For fly genetics, many binary transactivational systems have been exploited in vivo; however as the study of complex problems often requires multiple systems that can be used in parallel, there is a need to identify additional bipartite genetic systems. To expand this molecular genetic toolbox, we tested multiple bacterially-derived binary transactivational systems in Drosophila melanogaster including the p-CymR operon from Pseudomonas putida, PipR operon from Streptomyces coelicolor, TtgR operon from Pseudomonas putida, and the VanR operon from Caulobacter crescentus. Our work provides the first characterization of these systems in an animal model in vivo. For each system we demonstrate robust tissue-specific spatial transactivation of reporter gene expression, enabling future studies to exploit these transactivational systems for molecular genetic studies.

genetics↗

bioRxiv2019

The Developmental Transcriptome of Ae. albopictus, a Major Worldwide Human Disease Vector

Aedes albopictus mosquitoes are important vectors for a number of human pathogens including the Zika, dengue, and chikungunya viruses. Capable of displacing Aedes aegypti populations, it adapts to cooler environments which increases its geographical range and transmission potential. There are limited control strategies for Aedes albopictus mosquitoes which is likely attributed to the lack of comprehensive biological studies on this emerging vector. To fill this void, here using RNAseq we characterized Aedes albopictus mRNA expression profiles at 47 distinct time points throughout development providing the first high-resolution comprehensive view of the developmental transcriptome of this worldwide human disease vector. This enabled us to identify several patterns of shared gene expression among tissues as well as sex-specific expression patterns. Moreover, to illuminate the similarities and differences between Aedes aegypti, a related human disease vector, we performed a comparative analysis using the two developmental transcriptomes. We identify life stages were the two species exhibited significant differential expression among orthologs. These findings provide insights into the similarities and differences between Aedes albopictus and Aedes aegypti mosquito biology. In summary, the results generated from this study should form the basis for future investigations on the biology of Aedes albopictus mosquitoes and provide a goldmine resource for the development of transgene-based vector control strategies.

bioinformatics↗