Development of DNA-Based Lateral Flow Assay for Detection of LDLR Gene Mutation for Familial Hypercholesterolemia.

Background: The techniques for detecting single nucleotide polymorphisms (SNP) require lengthy and complex experimental procedures and expensive instruments that may only be available in some laboratories. Thus, a deoxyribonucleic acid (DNA)-based lateral flow assay (LFA) was developed as a point-of-care test (POCT) diagnostic tool for genotyping. In this study, single nucleotide variation (E101K) in the low-density lipoprotein receptor (LDLR) gene leading to familial hypercholesterolemia (FH) was chosen as a model. Methods: Hypercholesterolemic individuals (n = 103) were selected from the Malaysian Cohort project (UKM Medical Molecular Biology Institute) while the control samples were selected from the Biobank (UKM Medical Molecular Biology Institute). The DNA samples were isolated from whole blood. Polymerase chain reaction (PCR) amplification process was performed using bifunctional labelled primers specifically designed to correspond to the variant that differentiates wild-type and mutant DNA for visual detection on LFA. The variant was confirmed using Sanger sequencing, and the sensitivity and specificity of the LFA detection method were validated using the Agena MassARRAY® technique. Results: Out of 103 hypercholesterolemic individuals, 5 individuals (4.8%) tested positive for E101K, LDLR mutation and the rest, including healthy control individuals, tested negative. This result was concordant with Sanger sequencing and Agena MassARRAY®. These five individuals could be classified as Definite FH, as the DNA diagnosis was confirmed. The sensitivity and specificity of the variant detection by LFA is 100% compared to results using the genotyping method using Agena MassARRAY®. Conclusion: The developed LFA can potentially be used in the POC setting for detecting the E101K variant in the LDLR gene. This LFA can also be used to screen family members with E101K variant in the LDLR gene and is applicable for other SNP's detection.

Molecular survey of tick-borne infectious agents in cricetid rodents (Rodentia: Cricetidae) in Central and Southern Chile.

Tick-borne infectious agents (TBIAs) include several bacteria and protozoa that can infect vertebrates, including humans. Some of these agents can cause important diseases from both a public health perspective, such as Lyme disease, and from an animal health and production viewpoint, such as Texas fever. In Chile, several studies have assessed the presence of tick-borne disease agents in vectors and mammal hosts, mainly in the northern regions, but few studies have assessed the presence of these agents in Central and Southern Chile. This study aimed to assess the presence of three groups of TBIAs-Borrelia, Anaplasmataceae, and Piroplasmida-in cricetid rodents of Central and Southern Chile. A total of 207 specimens from 13 localities between the O'Higgins and Los Lagos regions were captured. DNA was extracted from the liver and spleen, and subsequently underwent polymerase chain reaction (PCR) amplification targeting the 16S rRNA, flaB, and 18S rRNA genes to detect DNA from Borrelia, Anaplasmataceae, and Piroplasmida, respectively. Although no DNA from these TBIAs was detected, the DNA extraction process was validated by optimal DNA purity ratios (an A260/A280 ratio within the 1.6-2.0 range) and successful internal control amplification in all samples. These results, in addition to findings from previous reports, suggest a very low prevalence of these TBIAs in the rodent population studied. Further research into the factors influencing the presence of these agents and their vectors will provide insight into the reasons underlying this low prevalence.

Mutagenic effects of a commercial glyphosate-based herbicide formulation on the soil filamentous fungus Aspergillus nidulans depending on the mode of exposure.

Glyphosate-based herbicides (GBH) are the most used pesticides worldwide. This widespread dissemination raises the question of non-target effects on a wide range of organisms, including soil micro-organisms. Despite a large body of scientific studies reporting the harmful effects of GBHs, the health and environmental safety of glyphosate and its commercial formulations remains controversial. In particular, contradictory results have been obtained on the possible genotoxicity of these herbicides depending on the organisms or biological systems tested, the modes and durations of exposure and the sensitivity of the detection technique used. We previously showed that the well-characterized soil filamentous fungus Aspergillus nidulans was highly affected by a commercial GBH formulation containing 450 g/L of glyphosate (R450), even when used at doses far below the agricultural application rate. In the present study, we analysed the possible mutagenicity of R450 in A. nidulans by screening for specific mutants after different modes of exposure to the herbicide. R450 was found to exert a mutagenic effect only after repeated exposure during growth on agar-medium, and depending on the metabolic status of the tested strain. The nature of some mutants and their ability to tolerate the herbicide better than did the wild-type strain suggested that their emergence may reflect an adaptive response of the fungus to offset the herbicide effects. The use of a non-selective molecular approach, the quantitative random amplified polymorphic DNA (RAPD-qPCR), showed that R450 could also exert a mutagenic effect after a one-shot overnight exposure during growth in liquid culture. However, this effect was subtle and no longer detectable when the fungus had previously been repeatedly exposed to the herbicide on a solid medium. This indicated an elevation of the sensitivity threshold of A. nidulans to the R450 mutagenicity, and thus confirmed the adaptive capacity of the fungus to the herbicide.

Drug discovery: Insights from the invertebrate Caenorhabditis elegans.

Therapeutic drug development is a long, expensive, and complex process that usually takes 12-15 years. In the early phases of drug discovery, in particular, there is a growing need for animal models that ensure the reduction in both cost and time. Caenorhabditis elegans has been traditionally used to address fundamental aspects of key biological processes, such as apoptosis, aging, and gene expression regulation. During the last decade, with the advent of large-scale platforms for screenings, this invertebrate has also emerged as an essential tool in the pharmaceutical research industry to identify novel drugs and drug targets. In this review, we discuss the reasons why C. elegans has been positioned as an outstanding cost-effective option for drug discovery, highlighting both the advantages and drawbacks of this model. Particular attention is paid to the suitability of this nematode in large-scale genetic and pharmacological screenings. High-throughput screenings in C. elegans have indeed contributed to the breakthrough of a wide variety of candidate compounds involved in extensive fields including neurodegeneration, pathogen infections and metabolic disorders. The versatility of this nematode, which enables its instrumentation as a model of human diseases, is another attribute also herein underscored. As illustrative examples, we discuss the utility of C. elegans models of both human neurodegenerative diseases and parasitic nematodes in the drug discovery industry. Summing up, this review aims to demonstrate the impact of C. elegans models on the drug discovery pipeline.

TrapSeq: An RNA Sequencing-Based Pipeline for the Identification of Gene-Trap Insertions in Mammalian Cells.

The development of haploid mammalian cell lines, coupled to next-generation sequencing, has recently facilitated forward genetic screenings in mammals. For mutagenesis, retrovirus- or transposon-based gene traps are frequently used. Current methods to map gene-trap insertions are based on inverse or splinkerette PCR, which despite their efficacy are prone to artifacts and do not provide information on expression of the targeted gene. Here, we describe a new RNA sequencing-based method (TrapSeq) to map gene-trap insertions. By recognizing chimeric mRNAs containing gene-trap sequences spliced to an exon, our method identifies insertions that lead to productive trapping. When applied to individual mutant clones, our method provides a fast and cost-effective way that not only identifies the insertion site but also reveals its impact on the expression of the trapped gene. As proof of principle, we conducted two independent screenings for resistance against 6-thioguanine and an ATR inhibitor, which identified mutations known to provide resistance to these reagents and revealed ECT2 as a novel determinant for the sensitivity to ATR inhibition.