Nucleic Acid Sequences Research Articles

Viral genome sequencing methods: benefits and pitfalls of current approaches.

Whole genome sequencing of viruses provides high-resolution molecular insights, enhancing our understanding of viral genome function and phylogeny. Beyond fundamental research, viral sequencing is increasingly vital for pathogen surveillance, epidemiology, and clinical applications. As sequencing methods rapidly evolve, the diversity of viral genomics applications and catalogued genomes continues to expand. Advances in long-read, single molecule, real-time sequencing methodologies present opportunities to sequence contiguous, haplotype resolved viral genomes in a range of research and applied settings. Here we present an overview of nucleic acid sequencing methods and their applications in studying viral genomes. We emphasise the advantages of different viral sequencing approaches, with a particular focus on the benefits of third-generation sequencing technologies in elucidating viral evolution, transmission networks, and pathogenesis.

Maximizing the Impact of Research Featuring Nucleic-Acid Sequencing Methods in Environmental Science & Technology and Environmental Science & Technology Letters.

Maximizing the Impact of Research Featuring Nucleic-Acid Sequencing Methods in Environmental Science & Technology and Environmental Science & Technology Letters.

Upregulation of EPSTI1/Drp1/AKT1 Signaling Pathways Using pDNA/Melittin Against Breast Cancer.

This study investigates the role of genes related to breast cancer in apoptosis control. A melittin nucleic acid sequence was synthesized and introduced into a pcDNA3.1(+) Mammalian Expression Plasmid. The cloning accuracy was assessed using PCR testing and enzyme digestion techniques. The vectors were transfected into cells using LipofectamineTM2000. The transfection efficacy of MCF-7 and 4T1 cells was evaluated using fluorescence and bright-field imaging. Pure melittin produced from bee venom had a notable hemolytic impact, with lower hemolytic activity levels than the positive control, Triton X-100. The growth rate of 4T1 and MCF-7 cancer cells was significantly inhibited. The apoptosis rates were 8.54%, 46.20%, and 78.82% for free pDNA, melittin, and pDNA-melittin, respectively. The C-pDNA/Melittin-treated group showed a statistically significant reduction in cancer factors compared to the control group. The treated tumors exhibited significant necrosis and late apoptosis, with a prevalence ranging from about 5% to 10% of the lesions. After exposure to pDNA-melittin, there was no significant increase in transcription levels of caspase-3, caspase-8, BCRA1, BAX, Drp1, AKT1, and EPSTI1 genes in the normal non-cancerous groups. The findings provide novel opportunities for the therapeutic targeting of malignancies via melittin and the stimulation of the EPSTI1/Drp1/AKT1 signaling cascades.

Next-Generation Sequencing and Emerging Technologies.

Genetic sequencing technologies are evolving at a rapid pace with major implications for research and clinical practice. In this review, the authors provide an updated overview of next-generation sequencing (NGS) and emerging methodologies. NGS has tremendously improved sequencing output while being more time and cost-efficient in comparison to Sanger sequencing. The authors describe short-read sequencing approaches, such as sequencing by synthesis, ion semiconductor sequencing, and nanoball sequencing. Third-generation long-read sequencing now promises to overcome many of the limitations of short-read sequencing, such as the ability to reliably resolve repeat sequences and large genomic rearrangements. By combining complementary methods with massively parallel DNA sequencing, a greater insight into the biological context of disease mechanisms is now possible. Emerging methodologies, such as advances in nanopore technology, in situ nucleic acid sequencing, and microscopy-based sequencing, will continue the rapid evolution of this area. These new technologies hold many potential applications for hematological disorders, with the promise of precision and personalized medical care in the future.

Design and Evaluation of a Robust CRISPR Kinetic Assay for Hot-Spot Genotyping.

Next-generation sequencing offers highly multiplexed and accurate detection of nucleic acid sequences but at the expense of complex workflows and high input requirements. The ease of use of CRISPR-Cas12 assays is attractive and may enable highly accurate detection of sequences implicated in, for example, cancer pathogenic variants. CRISPR assays often employ end-point measurements of Cas12 trans-cleavage activity after Cas12 activation by the target; however, end point-based methods can be limited in accuracy and robustness by arbitrary experimental choices. To overcome such limitations, we develop and demonstrate here an accurate assay targeting a mutation of the epidermal growth factor gene implicated in lung cancer (exon 19 deletion). The assay is based on characterizing the kinetics of Cas12 trans-cleavage to discriminate the mutant from wild-type targets. We performed extensive experiments (780 reactions) to calibrate key assay design parameters, including the guide RNA sequence, reporter sequence, reporter concentration, enzyme concentration, and DNA target type. Interestingly, we observed a competitive reaction between the target and reporter molecules that has important consequences for the design of CRISPR assays, which use preamplification to improve sensitivity. Finally, we demonstrate the assay on 18 tumor-extracted amplicons and 100 training iterations with 99% accuracy and discuss discrimination parameters and models to improve wild type versus mutant classification.

Development of recombinase polymerase amplification-based colorimetric detection assay for rapid identification of invasive cassava mealybug, Phenacoccus manihoti Matile-Ferrero

Phenacoccus manihoti Matile-Ferrero (Hemiptera: Pseudococcidae), is an economically important invasive cassava pest responsible for the massive devastation of cassava in Asia and African continent. Initially, identifying this invasive pest posed challenges because it closely resembled native mealybug species. Additionally, the traditional morphological identification process is labor-intensive and time-consuming. Detecting invasive pests at an early stage is crucial, hence development of a rapid detection assay is essential. In the current study, we have developed a simple, rapid, sensitive, and efficient molecular detection assay for P. manihoti based on Recombinase Polymerase Amplification (RPA). The primers for the RPA assay were designed using unique nucleic acid sequences of P. manihoti, and the protocol was standardized. Specificity test demonstrated that the RPA assay could amplify DNA of P. manihoti only, and no amplification was observed in six other mealybug species. The specificity of assay was confirmed using SYBR green-based colorimetric detection and gel electrophoresis where positive samples showed 195 bp amplicon size in P. manihoti samples. The assay successfully amplified P. manihoti DNA in thirty minutes at an annealing temperature of 41° C in a water bath and displayed a sensitivity of 72.5 picograms per microliter. The assay's simplicity, rapidity, and high sensitivity make it a valuable tool for detecting and monitoring P. manihoti in quarantine stations and facilitating in development of a portable diagnostic kit.

An Aptamer-Based Hydrogel Sensing Platform Enabling Low Micromolar Detection of Small Molecules Using Low Sample Volumes.

Poor fluorescence recovery at low analyte dosages and slow ligand binding kinetics are critical challenges currently limiting the use of aptamer-functionalized hydrogels for sensing small molecules. In this paper, we report an adenosine-responsive hydrogel sensor that integrates FRET-signaling aptamer switches into in situ-gelling thin-film hydrogels. The hydrogel sensor is able to entrap a high proportion of the sensing probes (>70% following vigorous washing), delay nucleolytic degradation, stabilize weak aptamer complexes to improve hybridization affinity and suppress fluorescence background, and provide high sensitivity in biological fluids (i.e., undiluted human serum). Furthermore, the developed hydrogel sensors were able to achieve low limits of detection (5.3 μM in buffer and 8.8 μM in serum) within 4 min of exposure to the sample, with signal generation requiring only 20 μL/well of analyte sample. The physical nature of the aptamer encapsulation allows this approach to accommodate virtually any small-molecule aptamer, avoiding the need for covalent anchoring and the complex modification of nucleic acid sequences typically required for effective aptamer-based molecular recognition.

Time-encoded electrical detection of trace RNA biomarker by integrating programmable molecular amplifier on chip

One of the serious challenges facing modern point-of-care (PoC) molecular diagnostic platforms relate to reliable detection of low concentration biomarkers such as nucleic acids or proteins in biological samples. Non-specific analyte-receptor interactions due to competitive binding in the presence of abundant molecules, inefficient mass transport and very low number of analyte molecules in sample volume, in general pose critical hurdles for successful implementation of such PoC platforms for clinical use. Focusing on these specific challenges, this work reports a unique PoC biosensor that combines the advantages of nanoscale biologically-sensitive field-effect transistor arrays (BioFET-arrays) realized in a wafer-scale top-down nanofabrication as high sensitivity electrical transducers with that of sophisticated molecular programs (MPs) customized for selective recognition of analyte miRNAs and amplification resulting in an overall augmentation of signal transduction strategy. The MPs realize a programmable universal molecular amplifier (PUMA) in fluidic matrix on chip and provide a biomarker-triggered exponential release of small nucleic acid sequences easily detected by receptor-modified BioFETs. A common miRNA biomarker LET7a was selected for successful demonstration of this novel biosensor, achieving limit of detection (LoD) down to 10 fM and wide dynamic ranges (10 pM–10 nM) in complex physiological solutions. As the determination of biomarker concentration is implemented by following the electrical signal related to analyte-triggered PUMA in time-domain instead of measuring the threshold shifts of BioFETs, and circumvents direct hybridization of biomarkers at transducer surface, this new strategy also allows for multiple usage (>3 times) of the biosensor platform suggesting exceptional cost-effectiveness for practical use.

A Sensitivity Study for Interpreting Nucleic Acid Sequence Screening Regulatory and Guidance Documentation: Toward a Foundational Synthetic Nucleic Acid Sequence Screening Framework

A Sensitivity Study for Interpreting Nucleic Acid Sequence Screening Regulatory and Guidance Documentation: Toward a Foundational Synthetic Nucleic Acid Sequence Screening Framework

Uncommon pathogen misidentification of Herbaspirillum huttiense as Burkholderia cepacia in bacteremia: a case report.

Herbaspirillum huttiense is an opportunistic pathogen associated with rare cases of bacteremia. In this case report, H huttiense was isolated from blood samples collected from an intravenous catheter (incubated for 20.8 hours) and a peripheral vein (incubated for 14.16 hours) of a lung adenocarcinoma patient. Positive blood culture bottles were subjected to smear preparation, and Gram staining and microscopic examination revealed the presence of gram-negative rods in both aerobic bottles. We used the VITEK MS automatic microbial mass spectrometry system, VITEK 2 Compact automatic microbial analysis system, and high-throughput nucleic acid sequencing for accurate identification of the isolate. It is noteworthy that although the VITEK 2 Compact identified the isolate as Burkholderia cepacia, confirmation through VITEK MS mass spectrometry and 16S ribosomal DNA (rDNA) sequencing identified it as H huttiense. Subsequently, antimicrobial susceptibility testing was performed using the broth microdilution method, following the guidelines for nonfermenting gram-negative bacilli provided by the Clinical and Laboratory Standards Institute. This case highlights the possibility of misidentification of H huttiense as B cepacia by VITEK 2 Compact in certain situations, emphasizing the importance of considering uncommon pathogens, such as H huttiense, in the context of bacteremia in cancer patients.

Abstract LB036: Back and forth from structure to functional target: The interplay between Vimentin and non-canonical nucleic acid arrangements

Abstract Vimentin, a member of type III intermediate filaments (IFs), is a cytoskeletal protein that covers multiple architectural and functional roles, including the maintenance of organelle positioning in the cytoplasm, the regulation of cytoskeletal rearrangements, while more recently its potential involvement as a regulatory factor in gene expression emerged. Additionally, it is involved in cell migration and epithelial-to-mesenchymal transition (EMT). In previous works, we identified Vimentin as the first known protein that selectively binds G-quadruplexes repeats (G4-repeats). This DNA folding consists of repeated units of nucleic acid sequences arranged into G-quadruplex (G4s). Their three-dimensional features largely differ from the most common single-stranded or double-helix nucleic acids as well as from the isolated G4 modules. Noteworthy, they occur at a small subset of genomic sites, associated with cell proliferation and migration, where they can recruit the soluble nuclear pool of Vimentin. Altogether, this information led us to consider the Vimentin-DNA complex as a promising target for developing small molecule binders capable of inhibiting cell migration and, eventually, EMT. To properly set up a rational drug-design approach, we integrated different advanced biophysical tools (Hydrogen-Deuterium Exchange, Cryo-Electron Microscopy) to derive a comprehensive picture of the protein-DNA complex at the molecular level. This approach allowed us to map the interaction surface between the soluble tetrameric form of Vimentin and a G4-repeat. Based on this data, we initiated a screening campaign using focused libraries of small molecules and peptides, which were rationally designed according to the structural features of the Vimentin-G4-repeats complex. The capability of the identified hits to prevent the recognition of the nucleic acid by Vimentin has been investigated This approach allowed us to identify attractive novel entities that prevent the Vimentin-G4-repeat complex formation according to different molecular mechanisms. These systems are now under further refinement moving from the test tube to the cellular environment, where the dynamic behavior of these nucleic acid domains as well as the recruitment of different proteins by Vimentin might alter the architectural features of the target.

Capsiate ameliorates secondary hyperparathyroidism by improving insulin sensitivity and inhibiting angiogenesis.

Secondary hyperparathyroidism has a significant impact on the overall well-being of the body. Capsiates, known for their antioxidant and metabolic properties, have emerged as a promising alternative treatment for secondary hyperparathyroidism. This study aims to evaluate the effects and mechanisms of capsiates in the treatment of secondary hyperparathyroidism. To achieve our research objectives, we conducted a study on patients' serum and examined changes in metabolic markers using serum metabolomics. We induced secondary hyperparathyroidism in rat through dietary intervention and divided them into four groups. The first group, referred to as the Parathyroid Hormone (PTH) group, received a low-calcium and high-phosphate diet (0.2% calcium, 1.2% phosphorus). The second group served as the control group, receiving a standard phosphate and calcium diet (0.6% calcium, 0.6% phosphorus). The third group, called the capsiates group, consisted of rat from the control group treated with capsiates (intraperitoneal injection of 2 mg/kg capsiates for 2 weeks after 2 weeks of dietary intervention). The fourth group was the capsiates-treated PTH group. Subsequently, we conducted ribose nucleic acid (RNA) sequencing on parathyroid gland cells and evaluated serum thyroxine levels, oxidative stress, expression of proteins associated with vascular neogenesis, measurement of SOD, GSH and 3-nitrotyrosine, micro-CT and histological staining. The serum metabolomic data revealed a significant decrease in capsiate levels in the secondary hyperparathyroidism group. Administration of capsiates to PTH rat resulted in increased calcium levels compared to the PTH group. Additionally, the PTH + Capsiates group showed significantly lower levels of PTH and phosphate compared to the PTH group. The PTH group exhibited a notable increase in the quantity and size of mitochondria compared to the control group. Following capsiates administration to the PTH group, there was a significant reduction in the number of mitochondria and length of microvilli, but an increase in the size of mitochondria compared to the PTH group. Sequencing analysis revealed that vascular endothelial growth factor (VEGF) and Vascular Endothelial Growth Factor Receptor 1 (VEGFR1) play crucial roles in this process. Vascular-related variables and downstream signalling were significantly elevated in hyperthyroidism and were alleviated with capsaicin treatment. Finally, combining capsiates with the PTH group improved bone mineral density, Tb.N, BV.TV, Cs.Th, Tt.Ar, OPG, Ob.TV and Oc.TV, as well as the mineral apposition rate, but significantly decreased Tb.Sp and Receptor Activator for Nuclear Factor-κ B Ligand (RANKL) compared to the PTH group. The findings suggest that capsiates can improve secondary hyperparathyroidism and ameliorated osteoporosis outcomes by inhibiting angiogenesis and reducing oxidative stress.

Application of CRISPR/Cas12a for SARS-CoV-2 Nucleic Acid Detection

SARS-CoV-2 spans 3 years, and although the virus has been adjusted from "Category B, Tube A" to "Category B, Tube B" it is still present in the environment, causing widespread epidemic infections and great impact on human health and the environment. Currently, there are various nucleic acid detection methods for this virus, and CRISPR methods are widely used to construct biosensors for accurate and rapid specific detection of nucleic acid sequences due to their powerful gene-editing capabilities. In this article, we introduce the application of different forms of sensors such as fluorescent paper strips built with CRISPR/Cas12a for SARS-CoV-2 nucleic acid detection.

TPMA: A two pointers meta-alignment tool to ensemble different multiple nucleic acid sequence alignments.

Accurate multiple sequence alignment (MSA) is imperative for the comprehensive analysis of biological sequences. However, a notable challenge arises as no single MSA tool consistently outperforms its counterparts across diverse datasets. Users often have to try multiple MSA tools to achieve optimal alignment results, which can be time-consuming and memory-intensive. While the overall accuracy of certain MSA results may be lower, there could be local regions with the highest alignment scores, prompting researchers to seek a tool capable of merging these locally optimal results from multiple initial alignments into a globally optimal alignment. In this study, we introduce Two Pointers Meta-Alignment (TPMA), a novel tool designed for the integration of nucleic acid sequence alignments. TPMA employs two pointers to partition the initial alignments into blocks containing identical sequence fragments. It selects blocks with the high sum of pairs (SP) scores to concatenate them into an alignment with an overall SP score superior to that of the initial alignments. Through tests on simulated and real datasets, the experimental results consistently demonstrate that TPMA outperforms M-Coffee in terms of aSP, Q, and total column (TC) scores across most datasets. Even in cases where TPMA's scores are comparable to M-Coffee, TPMA exhibits significantly lower running time and memory consumption. Furthermore, we comprehensively assessed all the MSA tools used in the experiments, considering accuracy, time, and memory consumption. We propose accurate and fast combination strategies for small and large datasets, which streamline the user tool selection process and facilitate large-scale dataset integration. The dataset and source code of TPMA are available on GitHub (https://github.com/malabz/TPMA).

Artificial nanochannels for highly selective detection of miRNA based on the HCR signal amplification

Artificial solid-state nanochannels have nanoscale spatial confinement and unique ion transport properties that have been widely used in biosensing, nanofluidic devices, and nucleic acid sequencing. However, most of the nanochannel sensors for miRNA detection are grounded on the complementary pairing of bases, lacking signal amplification, which limits its application in many fields. Herein, we proposed a signal amplification strategy for miRNA-182 detection based on introducing hybridization chain reaction (HCR) in artificial nanochannels to improve the detection sensitivity. In this method, the capture DNA (cDNA) was anchored in the nanochannel, and when the target miRNA appeared, the hairpin probes H1, H2, and initiator were introduced to trigger the HCR reaction to form long nucleic acid double strands, which significantly enhanced the negative charge and affected ion transport in nanochannel. The nanochannel sensor performed excellent selectivity to the target miRNA-182, and it can realize the detection of miRNA-182 with an ultra-low concentration of 1.65 aM, which can be further applied to the detection of miRNA-182 in actual biological samples. Moreover, the nanochannel system was proved by using the AND logic gate that cDNA, initiator, H1, H2, and miRNA-182 are indispensable for successfully triggering HCR. This work provides a simple and efficient signal amplification method for miRNA detection, which considerably expands the application of nanochannel sensors and holds great prospects in biomedical and clinical detection.

Development of a LAMP-Based Diagnostic for the Detection of Multiple HIV-1 Strains.

Since its first appearance in 1981, HIV-1 has remained a global concern. Current methods for diagnosing HIV-1, while effective, are mostly specific to a given subtype of HIV-1 and often require expensive equipment and highly trained individuals to collect and process the sample. It is necessary to develop a sensitive diagnostic method that can be administered with minimal equipment to provide better care in low-resource settings. Loop-mediated isothermal amplification is a rapid and sensitive method for detecting the presence of specific nucleic acid sequences. Herein we report the development and comparison of two different HIV LAMP assays, integrase and VPR, as well as the comparison between TRIZol and magnetic beads RNA extraction methods for each assay. Our analysis shows that the integrase assay was able to detect the virus from multiple subtypes in under 30 min with a variable limit of detection (LOD) that was dependent on the HIV-1 subtype.

High-throughput homogenous assay for the direct detection of Listeria monocytogenes DNA

The Amplified Luminescent Proximity Homogenous Assay-linked Immunosorbent Assay (AlphaLISA) is known for detecting various protein targets; however, its ability to detect nucleic acid sequences is not well established. Here, the capabilities of the AlphaLISA technology were expanded to include direct detection of DNA (aka: oligo-Alpha) and was applied to the detection of Listeria monocytogenes. Parameters were defined that allowed the newly developed oligo-Alpha to differentiate L. monocytogenes from other Listeria species through the use of only a single nucleotide polymorphism within the 16S rDNA region. Investigations into the applicability of this assay with different matrices demonstrated its utility in both milk and juice. One remarkable feature of the oligo-Alpha is that greater sensitivity could be achieved through the use of multiple acceptor oligos compared to only a single acceptor oligo, even when only a single donor oligo was employed. Additional acceptor oligos were easily incorporated into the assay and a tenfold change in the detection limit was readily achieved, with detection limits of 250 attomole of target being recorded. In summary, replacement of antibodies with oligonucleotides allows us to take advantage of genotypic difference(s), which both expands its repertoire of biological markers and furthers its use as a diagnostic tool.

Abstract 3671: Analytical performance of the Oncomine™ Dx Express Test, a CE-IVD NGS liquid biopsy assay for identification of clinically relevant variants

Abstract For in vitro Diagnostic use only. Not available in all regions including the United States. Introduction Minimally invasive blood-based liquid biopsy using cell-free total nucleic acid (cfTNA) and next generation sequencing (NGS) has substantially evolved in the field of clinical diagnostics for oncology, for detection of molecular therapeutic targets in non-small cell lung cancer (NSCLC). Here we describe analytical validation results for cfTNA using the CE-IVD Oncomine™ Dx Express Test (ODxET) assay, a qualitative in vitro diagnostic test that uses targeted NGS technology, the Ion Torrent Genexus™ Dx Integrated Sequencer to detect SNV, indel and copy number gain present in 42 genes and fusions in 7 genes from cfTNA isolated from NSCLC plasma samples. Methods Plasma samples from NSCLC patients were screened for variants of interest on several important gene loci. Screening involved isolation of cfTNA from K2-EDTA-derived plasma using the Genexus™ Cell-Free Total Nucleic Acid Purification kit on an automated purification system and sequencing was performed on the Genexus™ Dx integrated sequencer using the ODxET assay. Plasma samples having clinically relevant variants from the set of screened plasma samples were used to perform the validation studies. Results Analytical sensitivity and specificity of NSCLC plasma samples were characterized through determination of limit of detection (LOD) and limit of blank (LOB), respectively. The LOD level tested for DNA SNVs, and indels at 5 ng (minimum) input level ranged from 0.62% to 1.82% allelic frequency (AF), while 30 ng (maximum) input shows the LOD level ranging from 0.23% to 0.42% AF. The LOD for the tested RNA fusions ranged from 4.2 to 19.6 molecular counts for 5ng input and 4.9 to 8.0 molecular counts for 30ng input. Furthermore, LOB for the ODxET assay was determined by testing wild type (WT) cfTNA extracted from 30 healthy donor blood plasma samples, which were confirmed to be negative at all variant locations. The analytical accuracy study shows the false positive rate at 0.20% for SNVs, 0% for indel locations and fusion targets. In addition, the analytical reproducibility study shows 99.64% intra-run repeatability call rate for DNA variants and 98.75% for RNA variants. Conclusion In conclusion, we establish that the ODxET assay on Ion Torrent Genexus Dx Integrated Sequencer, is fast and efficient while demonstrating high sensitivity, specificity, and reproducibility for testing NSCLC liquid biopsy samples. Citation Format: Madhuri Jasti, Swasti Raut, Diarra Hassell, Nader Ezzedine, Daniela Garcia, Stephen Wunsch, Nick Siepert, Elliott Martinez, Emilia Ostrowska, Jeff Schagemann, Jian Gu, Thilanka Jayaweera, Luming He, Angie Cheng. Analytical performance of the Oncomine™ Dx Express Test, a CE-IVD NGS liquid biopsy assay for identification of clinically relevant variants [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3671.

Molecular epidemiology of foot-and-mouth disease (review)

Molecular epidemiological studies are an important tool for regional and global surveillance of foot-and-mouth disease (FMD). These tests are based on constantly progressing technologies of nucleic acid sequencing and phylogenetic analysis. The use of these technologies made it possible to assess the genetic diversity of the FMD virus, to analyze the evolution of the virus in the enzootic regions, and track the pathways of FMD epizootic and panzootic spread. Molecular epidemiological studies have shown that within the long-known seven serotypes of the FMD virus, there are numerous topotypes (geographical types), genetic lineages and sublineages. Usually, the foot-and-mouth disease virus of a certain topotype and genetic lineage evolves within a certain area, periodically causing regional epizootics. However, over the past 30 years, two FMD panzootics have occurred, invloving several continents. The first panzootic occurred in the late 1990s – early 2000s and was caused by O/ME-SA/PanAsia FMDV, and the second, caused by O/ME-SA/Ind-2001 virus, began in 2013 and continues to the present. The emergence of FMD panzootics is probably a consequence of the economic globalization. FMD is not enzootic in Russia, but sporadic outbreaks of this disease are periodically reported. Molecular epidemiological studies have shown that these outbreaks are caused by the infection introduction from neighboring Asian countries, mainly from China. The FMD virus, which has come to the Russian Federation from other countries, is characterized by great genetic diversity and belongs to three serotypes, five topotypes and eight genetic lineages: O/Cathay, O/ME-SA/PanAsia, O/SEA/Mya-98, O/ME-SA/Ind-2001, O/ME-SA/unnamed, A/Asia/Iran-05, A/Asia/Sea-97, Asia1/V. The results of molecular epidemiological studies are taken into account when vaccine strains are to be selected for preventive vaccination of livestock in FMD high-risk areas. The review is based on the analysis of 68 literature sources.

Abstract 5922: Characterization of the biological effects of a quadruplex-interacting molecule in dedifferentiated liposarcoma cells

Abstract G-rich nucleic acid sequences can assume, under physiologic conditions, non-B secondary structures called G-quadruplexes (G4). The human genome contains thousands of putative quadruplex forming sequences (PQS), of which telomeres are the most extensively studied. PQS are frequently found in the promoters of oncogenes and transcription factors rather than in tumor suppressor or housekeeping genes, indicating an evolutionary selection of these elements based on gene function and suggesting a pivotal role of G4 in the regulation of gene expression. These observations have provided the rationale to explore G4 targeting as a therapeutic approach in cancer. Several small molecules have been indeed designed and characterized as G4-ligands (G4-L), most of them showing a “multiple-target” binding modality. Dedifferentiated liposarcoma (DDLPS) is a non-lipogenic disease characterized by aggressive local growth and multifocal local recurrences and the ability to elicit distant metastases. At the molecular level, DDLPS is marked by the amplification of the 12q13-q15 chromosomal region, leading to the abnormal expression of the MDM2 oncogene. The gene has emerged as an actionable therapeutic target and, recently, PQS within its promoter have been reported to fold into G4 structures. In addition, in our previous study, we observed that pathways related to telomere maintenance are remarkably up-modulated in DDLPS. These findings open a new avenue to explore the therapeutic potential of G4-L in this malignancy. In the present study, the biological activity of a tetra-substituted naphthalene diimide (NDI) G4-L derivative (SOP1812/QN-302) has been characterized in two patient-derived DDLPS cell lines. In particular, the exposure of DDLPS cells to increasing concentrations of SOP1812 resulted in a marked cytotoxic activity in vitro, with IC50 values within the sub-micromolar range (~0.3 μM). Moreover, DDLPS cells treated with equitoxic amounts of the compound were characterized by an 80-90% inhibition of telomerase activity and a remarkable perturbation in the MDM2-p53 feedback loop, leading to a pronounced accumulation of basal and phosphorylated (S15/S46/S392) p53 protein. These effects were paralleled by a 3-4 time increase in the number of γ-H2AX foci, the appearance of the cleaved form of PARP-1 and a pronounced inhibition of cell growth over time. Altogether, our findings indicate that targeting G4 structures by quadruplex-interacting molecules may represent a potential novel and effective therapeutic strategy in DDLPS. Citation Format: Eisa Naghshineh, Beatrice Tosoni, Lorenzo Di Pietro, Sandro Pasquali, Nadia Zaffaroni, Stephen Neidle, Sara N. Richter, Marco Folini. Characterization of the biological effects of a quadruplex-interacting molecule in dedifferentiated liposarcoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5922.