Nucleic Acid Sequences Research Articles

Pathogen dynamics and discovery of novel viruses and enzymes by deep nucleic acid sequencing of wastewater

Wastewater contains an extensive reservoir of genetic information, yet largely unexplored. Here, we analyzed by high-throughput sequencing total nucleic acids extracted from wastewater samples collected during a 17 month-period in Berlin, Germany. By integrating global wastewater datasets and applying a novel computational approach to accurately identify viral strains within sewage RNA-sequencing data, we demonstrated the emergence and global dissemination of a specific astrovirus strain. Astrovirus abundance and sequence variation mirrored temporal and spatial patterns of infection, potentially serving as footprints of specific timeframes and geographical locations. Additionally, we revealed more than 100,000 sequence contigs likely originating from novel viral species, exhibiting distinct profiles in total RNA and DNA datasets and including undescribed bunyaviruses and parvoviruses. Finally, we identified thousands of new CRISPR-associated protein sequences, including Transposase B (TnpB), a class of compact, RNA-guided DNA editing enzymes. Collectively, our findings underscore the potential of high-throughput sequencing of total nucleic acids derived from wastewater for a broad range of applications.

Point-of-Care Molecular Testing with tinto rangTM: A Food Grade Safe Fluorophore for Colorimetric LAMP Assays at Low Resource Settings

LAMP (loop-mediated isothermal amplification) has proven to be a highly robust, sensitive, and cost-effective method for detecting specific target nucleic acid sequences in a single-tube reaction without the need for sophisticated instruments. Traditionally, pH-sensitive dyes, DNA-binding dyes like Ethidium Bromide(EtBr), and SYBR have been employed to detect amplified products. However, these dyes present several drawbacks, limiting the widespread adoption of LAMP in diagnostic applications. To address this limitation, a novel dye called tinto rangTM has been introduced with promising applications in nucleic acid analysis. In this study, we present an evaluation of tinto rangTM's performance in comparison to commercially available dyes commonly used in LAMP assays. One of the unique advantages of tinto rangTM is its capability to facilitate the analysis of amplified nucleic acid products through three distinct methods: visual color change, changes in relative fluorescence, and turbidity. In addition to assessing tinto rangTM's performance, we also investigated various approaches to tackle the issue of non-specific amplification, which has been a common challenge in LAMP reactions. The experimental results unequivocally demonstrate that tinto rangTM outperforms pH-indicating and other DNA-binding dyes used in LAMP assays, making it a superior alternative. This study paves the way for the broader adoption of tinto rangTM in various applications, including point-of-care diagnostics, disease monitoring, and molecular biology research.

The non-negligible non-specific adsorption of oligonucleotides in target-immobilized Mag-SELEX

Target-immobilized magnetic beads-based Systematic Evolution of Ligands by Exponential Enrichment (target-immobilized Mag-SELEX) has emerged as a powerful tool for aptamer selection owing to its convenience, efficiency, and versatility. However, in this study we systematically investigated non-specific adsorption in target-immobilized Mag-SELEX and found that the non-specific adsorption of the oligonucleotides to target-labeled magnetic beads was comparable to that of the screening libraries, indicating a substantial portion of captured sequences likely stem from non-specific adsorption. Longer nucleic acid sequences (80 nt and above, such as polyA80 and yeast tRNA) were found to attenuate this non-specific adsorption, with more complex higher-order structures demonstrating greater efficacy, while dNTP and short sequences such as primer sequences (20 nt), polyT(59), or polyA(59), did not possess this capability. Various evidence suggested that hydrophobic interactions and other weak interactions may be the primary underlying cause of non-specific adsorption. Additionally, surface modification of magnetic beads with polar molecule polyethylene glycol (PEG) also yielded a significant reduction in non-specific adsorption. In conclusion, our research underscores the critical importance of closely monitoring non-specific adsorption in target-immobilized Mag-SELEX.

CRISPR/Cas12a-Based APE1 Enzyme Cleavage Assay for Drug Resistance Analysis of Staphylococcus aureus-Related Pneumonia.

Drug resistance analysis of Staphylococcus aureus is responsible for generating significant mortality and morbidity in numerous diseases. However, sensitive and accurate analysis of drug resistance of S. aureus remains a huge challenge. In this study, we present the development of a fluorescence biosensor based on the CRISPR/Cas12a system that enables label-free and ultrasensitive detection of the mecA gene in methicillin-resistant S. aureus (MRSA). The biosensor identified the mecA gene in MRSA using Cas12a/crRNA. This recognition triggered the trans-cleavage activity of Cas12a and the release of RNA1, which subsequently induced Apurinic/apyrimidinic endonuclease 1 (APE1) enzyme-assisted target recycling and G-quadruplexes/Thioflavin T-based signal reaction. Based on this, the biosensor effectively detects the mecA gene with a low limit of detection of 212 aM and a high degree of selectivity, even toward single base mutations. Compared with the traditional CRISPR-Cas12a system-based methods, in which the signal amplification process is prone to generate nucleic acid sequence mismatch, which causes errors, the biosensor used APE1 to improve nucleic acid sequence recognition specificity to ensure that the RNA1 sequence released after Cas12a/crRNA cleavage can specifically guide the signal cycle. In addition to enhancing the CRISPR toolkit, the developed biosensor offers a novel method for the precise and sensitive identification of drug-resistant microbes that cause infections.

A Review of Next-Generation Sequencing Technologies and Their Impact on Clinical Research: Assessing Clinical Efficacy and Cost-Effectiveness

In contrast to microarray methods, sequence-based technologies directly determine the nucleic acid sequence. A number of modern sequencing technologies are referred to collectively as "next-generation sequencing" (NGS), often known as "high-throughput sequencing." Compared to conventional Sanger sequencing, NGS gives orders of magnitude more data at a much lower ongoing cost. These new technologies allow for much faster and more affordable sequencing of DNA and RNA, revolutionizing the study of genomics and molecular biology. Technical improvements in NGS sequencing methods have rapidly increased sequencing volume to several billion nucleotides within a short period and at a reasonable cost. Currently, NGS is developing into a molecular microscope that is permeating almost all areas of biological research. The last ten years have seen the development of NGS platforms and methodologies, and the quality of the sequences has increased to the point where NGS is now utilized in human clinical diagnosis. Due to significant cost reductions and greater community acceptance of NGS, the utilization of NGS techniques in studying clinical trials has significantly increased. NGS is a useful tool for detecting mutations in people with cancer and genetic abnormalities. To ascertain whether NGS can cost-effectively improve patient outcomes, more thorough cost-effectiveness studies of NGS applied to patient care management are required.

Genetic alterations in the pmrAB two-component system and lipid A biosynthesis genes of polymyxin-resistant Acinetobacter baumannii isolates.

The rate of pandrug-resistant Acinetobacter baumannii strains is on the rise in all continents. This bacterium can acquire resistance to all antibiotics, even to colistin. Alterations in the lipid A or/and the two-component pmrAB were earlier detected in colistin resistance. We investigated and analyzed two strains of A. baumannii (ABRC1 and ABRC2) isolated from two patients admitted to intensive care unit with a septic shock. Both strains were resistant to all tested antibiotics including colistin with a MIC >256 mg L-1. Colistin resistance genes (pmrA, pmrB, lpxA, lpxC, lpxD, and lpsB) of two strains (ABRC1 and ABRC2) were investigated by PCR and sequencing. Obtained nucleic acid sequences were aligned with reference sequences of ATCC 19606 and 17987. In this study two amino acid mutations, N287D in the lpxC gene and E117K in the lpxD gene, were detected in both ABRC1 and ABRC2 strains. ABRC1 had an additional H200L mutation in the pmrA gene. Both colistin resistant strains harbored the same A138T mutation in the pmrB gene. The ABRC2 strain also had an alteration in the kinase domain, specifically an R263S substitution of the histidine kinase domain. Three identical mutations were found in the lpsB gene of both A. baumannii strains: Q216K + H218G + S219E. As a result, a newly deduced protein sequence in both ABRC1 and ABRC2 strains differed from those described in ATCC 17978 and 19606 strains was determined. Colistin resistance is multifactorial in A. baumannii. In our study we detected novel mutations in colistin resistant A. baumannii clinical isolates.

Amplifiable protein identification via residue-resolved barcoding and composition code counting.

Ultrasensitive protein identification is of paramount importance in basic research and clinical diagnostics but remains extremely challenging. A key bottleneck in preventing single-molecule protein sequencing is that, unlike the revolutionary nucleic acid sequencing methods that rely on the polymerase chain reaction (PCR) to amplify DNA and RNA molecules, protein molecules cannot be directly amplified. Decoding the proteins via amplification of certain fingerprints rather than the intact protein sequence thus represents an appealing alternative choice to address this formidable challenge. Herein, we report a proof-of-concept method that relies on residue-resolved DNA barcoding and composition code counting for amplifiable protein fingerprinting (AmproCode). In AmproCode, selective types of residues on peptides or proteins are chemically labeled with a DNA barcode, which can be amplified and quantified via quantitative PCR. The operation generates a relative ratio as the residue-resolved 'composition code' for each target protein that can be utilized as the fingerprint to determine its identity from the proteome database. We developed a database searching algorithm and applied it to assess the coverage of the whole proteome and secretome via computational simulations, proving the theoretical feasibility of AmproCode. We then designed the residue-specific DNA barcoding and amplification workflow, and identified different synthetic model peptides found in the secretome at as low as the fmol/L level for demonstration. These results build the foundation for an unprecedented amplifiable protein fingerprinting method. We believe that, in the future, AmproCode could ultimately realize single-molecule amplifiable identification of trace complex samples without further purification, and it may open a new avenue in the development of next-generation protein sequencing techniques.

Metatranscriptomic analysis of common mosquito vector species in the Canadian Prairies.

Mosquitoes are the most dangerous animals on the planet, responsible for over 800,000 deaths per year globally. This is because they carry and transmit a plethora of human disease-causing microorganisms, such as West Nile virus and the malaria parasite. Recent innovations in nucleic acid sequencing technologies have enabled researchers unparalleled opportunities to characterize the suite of microorganisms harbored by different mosquito species, including the causal agents of disease. In our study, we carried out 3 years of intensive mosquito surveillance in Canada. We collected and characterized the microorganisms harbored by >35,000 mosquitoes, including the identification of the agents of Cache Valley fever, avian malaria, and canine heartworm. We also detected insect-specific viruses and discovered 17 new viruses that have never been reported. This study, which is the first of its kind in Canada and one of only a handful globally, will greatly aid in future infectious disease research.

Screening and identification of DNA nucleic acid aptamers against F1 protein of Yersinia pestis using SELEX method.

Yersinia pestis is a bacterium that causes the disease plague. It has caused the deaths of many people throughout history. The bacterium possesses several virulence factors (pPla, pFra, and PYV). PFra plasmid encodes fraction 1 (F1) capsular antigen. F1 protein protects the bacterium against host immune cells through phagocytosis process. This protein is specific for Y. pestis. Many diagnostic techniques are based on molecular and serological detection and quantification of F1 protein in different food and clinical samples. Aptamers are small nucleic acid sequences that can act as specific ligands for many targets.This study, aimed to isolate the high-affinity ssDNA aptamers against F1 protein. In this study, SELEX was used as the main strategy in screening aptamers. Moreover, enzyme-linked aptamer sorbent assay (ELASA) and surface plasmon resonance (SPR) were used to determine the affinity and specificity of obtained aptamers to F1 protein. The analysis showed that among the obtained aptamers, the three aptamers of Yer 21, Yer 24, and Yer 25 were selected with a KD value of 1.344E-7, 2.004E-8, and 1.68E-8M, respectively. The limit of detection (LoD) was found to be 0.05, 0.076, and 0.033μg/ml for Yer 21, Yer 24, and Yer 25, respectively. This study demonstrated that the synthesized aptamers could serve as effective tools for detecting and analyzing the F1 protein, indicating their potential value in future diagnostic applications.

Mechanisms and Delivery of tRNA Therapeutics.

Transfer ribonucleic acid (tRNA) therapeutics will provide personalized and mutation specific medicines to treat human genetic diseases for which no cures currently exist. The tRNAs are a family of adaptor molecules that interpret the nucleic acid sequences in our genes into the amino acid sequences of proteins that dictate cell function. Humans encode more than 600 tRNA genes. Interestingly, even healthy individuals contain some mutant tRNAs that make mistakes. Missense suppressor tRNAs insert the wrong amino acid in proteins, and nonsense suppressor tRNAs read through premature stop signals to generate full length proteins. Mutations that underlie many human diseases, including neurodegenerative diseases, cancers, and diverse rare genetic disorders, result from missense or nonsense mutations. Thus, specific tRNA variants can be strategically deployed as therapeutic agents to correct genetic defects. We review the mechanisms of tRNA therapeutic activity, the nature of the therapeutic window for nonsense and missense suppression as well as wild-type tRNA supplementation. We discuss the challenges and promises of delivering tRNAs as synthetic RNAs or as gene therapies. Together, tRNA medicines will provide novel treatments for common and rare genetic diseases in humans.

Integration of a Cas12a-mediated DNAzyme actuator with efficient RNA extraction for ultrasensitive colorimetric detection of viral RNA

Developing highly sensitive and specific on-site tests is imperative to strengthen preparedness against future emerging infectious diseases. Here, we describe the construction of a Cas12a-mediated DNAzyme actuator capable of converting the recognition of a specific DNA sequence into an amplified colorimetric signal. To address viral RNA extraction challenges for on-site applications, we developed a rapid and efficient method capable of lysing the viral particles, preserving the released viral RNA, and concentrating the viral RNA. Integration of the DNAzyme actuator with the viral RNA extraction method and loop-mediated isothermal amplification enables a streamlined colorimetric assay for highly sensitive colorimetric detection of respiratory RNA viruses in gargle and saliva. This assay can detect as few as 83 viral particles/100 μL in gargle and 166 viral particles/100 μL in saliva. The entire assay, from sample processing to visual detection, was completed within 1 h at a single controlled temperature. We validated the assay by detecting SARS-CoV-2 in 207 gargle and saliva samples, achieving a clinical sensitivity of 96.3 % and specificity of 100%. The assay is adaptable for detecting specific nucleic acid sequences in other pathogens and is suitable for resource-limited settings.

Modern trends in the application of thermoelectric method in non-destructive testing (review)

The article provides an overview of the main directions of using the thermoelectric testing method in various spheres of the national economy. The thermoelectric method is most widely used in industry. There are publications on the application of the thermoelectric method for quality monitoring of turning and friction stir welding. It is shown that the thermoelectric method makes it possible to increase the reliability of testing and, based on testing data, ensure optimal modes of the technological process of metalworking and welding. A number of articles are devoted to the application of the thermoelectric method to plastic deformation testing, the dependence of thermal EMF on the degree of plastic deformation is revealed. Recently, publications have appeared on the application of the thermoelectric method to testing the thermal resistance of the design “housing of a power semiconductor device-thermal interface-cooling radiator”. This design is very widespread in electronic equipment. In addition, there are articles on the use of the thermoelectric method to testing the transient resistance of contacts in the power supply network. It is shown that with an increase in contact resistance, the value of thermal EMF increases proportionally, which can be used to prevent emergencies in the power supply network. The thermoelectric method has also been successfully applied to diagnose the degree of titanium flooding. The dependence of the thermal EMF value on the degree of hydrogenation has been revealed. The use of the thermoelectric method is not limited to the field of industrial production. The thermoelectric method is successfully used in medicine for the undetectable detection of nucleic acid sequences, for the temperature diagnosis of human teeth, as well as for the diagnosis of inflammatory processes in the human body.

Dermaseptin B2 bioactive gene's potential for anticancer and anti-proliferative effect is linked to the regulation of the BAX/BBC3/AKT pathway.

Dermaseptin B2 (DrsB2) is an antimicrobial peptide with anticancer and angiostatic properties. We aimed to assess the in vitro inhibitory effect of pDNA/DrsB2 on the growth of breast cancer cells and its impact on the expression of genes involved in the BAX/BBC3/AKT pathway. The nucleic acid sequence of DrsB2 was artificially synthesized and inserted into the pcDNA3.1( +) Mammalian Expression Plasmid. PCR testing and enzyme digesting procedures evaluated the accuracy of cloning. The vectors were introduced into cells using LipofectamineTM2000 transfection reagent. The breast cancer cells were assessed by flow cytometry, MTT assessment, soft agar colony method, and wound healing investigation. The gene's transcription was evaluated using real-time PCR with a significance level of P < 0.05. The recombinant plasmid harboring the pDNA/DrsB2 vector was effectively produced, and the gene sequence showed absolute homogeneity (100% similarity) with the DrsB2 gene. The transfection effectiveness of MCF-7 and MCF-10A cells was 79% and 68%, respectively. The findings are measured using the growth inhibition 50% (GI50) metric, which indicates the concentration of pDNA/DrsB2 that stops 50% of cell growth. The proportions of early apoptosis, late apoptosis, necrosis, and viable MCF-7 cells in the pDNA/DrsB2 group were 40.50%, 2.31%, 1.69%, and 55.50%, respectively. The results showed a 100% increase in gene expression in programmed cell death following treatment with pDNA/DrsB2 (**P < 0.01). To summarize, the results described in this work offer new possibilities for treating cancer by targeting malignancies via pDNA/DrsB2 and activating the BAX/BBC3/AKT signaling pathways.

Sequencing is a Technological Part in Obtaining Primary Data on Nucleic Acid Sequences: Features to be Taken into Account when Performing it to Avoid Errors in the Test Procedure

Sequencing is a Technological Part in Obtaining Primary Data on Nucleic Acid Sequences: Features to be Taken into Account when Performing it to Avoid Errors in the Test Procedure

A novel syndrome of silent rhinovirus-associated bronchoalveolitis in children with recurrent wheeze

BackgroundRhinovirus (RV) infections trigger wheeze episodes in children. Thus, understanding of the lung inflammatory response to RV in children with wheeze is important. ObjectiveTo examine the associations of RV on lung lavage (BAL) granulocyte patterns and biomarkers of inflammation with age in children with treatment-refractory, recurrent wheeze (n=616). MethodsChildren underwent BAL to examine viral nucleic acid sequences, bacterial cultures, granulocyte counts, and phlebotomy for both general and type-2 inflammatory markers. ResultsDespite the absence of cold symptoms, RV was the most common pathogen detected (30%), and when present, was accompanied by BAL granulocytosis in 75% of children. Compared to children with no BAL pathogens (n=341), those with RV alone (n=127) had greater (p < 0.05) isolated neutrophilia (43% versus 16%), mixed eosinophils and neutrophils (26% versus 11%), and less pauci-granulocytic (27% versus 61%) BAL. Children with RV alone furthermore had biomarkers of active infection with higher total blood neutrophils and serum c-reactive protein (CRP), but no differences in blood eosinophils or total IgE. With advancing age, the log odds of BAL RV alone were lower, 0.82 [0.76-0.88, p < 0.001], but higher, 1.58 [1.01-2.51, p = 0.04], with high-dose daily corticosteroid treatment. ConclusionsChildren with severe recurrent wheeze often (22%) have a silent syndrome of lung RV infection with granulocytic bronchoalveolitis and elevated systemic markers of inflammation. The syndrome is less prevalent by school age, and not informed by markers of type-2 inflammation. We speculate that dysregulated mucosal innate antiviral immunity is a responsible mechanism.

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.

Progress and Prospects for a Nucleic Acid Screening Test Set.

DNA synthesis companies screen orders to detect controlled sequences with misuse risks. Assessing screening accuracy is challenging owing to the breadth of biological risks and ambiguities in risk definitions. Here, we detail an International Gene Synthesis Consortium working group's rationale and process to develop a prototype DNA synthesis screening test dataset, aiming to establish a baseline of screening system accuracy to compare with various screening approaches. Construction of the prototype test dataset involved four tool developers screening nucleic acid sequences from three taxonomic clusters of controlled organisms (Orbivirus, Francisella tularensis, and Coccidioides). Results were mapped onto predefined, comparable categories, checking for consensus or conflicts. Conflicts were grouped based on gene annotation and resolved through discussion. The process highlighted several long-standing challenges in DNA synthesis screening, including the qualitative differences in approaches taken by screening tools. Our findings highlight the lack of clarity in assessing pathogen sequences with respect to regulatory control language, compounded by scientific uncertainty. We illustrate the current degree of consensus and existing challenges using classification statistics and specific examples. This prototype underscores the necessity of expert-regulator coordination in assessing gene-associated risks, offering a template for creating test sets across all taxonomic groups on international control lists. Expanding the working group would enrich dataset comprehensiveness, enabling a transition from species-focused to function-focused regulatory controls. This sets the foundation for quality control, certification, and improved risk assessment in DNA synthesis screening.

Abstract 1120: Untargeted 16s Ribosomal Rna Survey Of Microbial Communities In The Patient With Aneurysmal And Occlusive Aortic Disease

Introduction: Inflammation is a hallmark of aneurysmal disease, but the etiology for the inflammation is unknown. Previously, bacteria and bacterial nucleic acid sequences have been identified within abdominal aortic aneurysm tissue. However, only one study has taken an un-targeted approach to assess the diversity of species and no study has surveyed bacterial species across the various environments in patients with aortic disease. Research Question: Does the distribution of bacterial sequences differ between environments and by disease status in patients with abdominal aortic disease? Methods: Whole blood, acute (luminal) thrombus, chronic (medial/abluminal) thrombus, and aortic wall specimens were obtained from 14 patients undergoing open aortic surgery. Five patients had aneurysm disease and nine patients had aortic occlusive disease. Sequencing using universal primers targeting the V3 - V5 16s rRNA bacterial gene was performed. Reads were processed and aligned to reference banks using QIIME2 to the genus level. Results: The average patient age was 60.9 years with no significant difference in baseline demographics. Phyllobacterium was the most common genus identified in acute and chronic thrombus for aneurysmal and occlusive disease while pseudomonas was the most common genus identified in aortic wall. Chao1 alpha diversity was higher in aneurysmal aortic wall samples compared to occlusive disease (10.6 vs 6.6, p=0.047). On principle component analysis, samples from similar tissue environments cluster together with 16% of the sequence variation explained by the grouping (p=0.003, Figure 1). Conclusions: The diversity of bacterial sequences groups by microenvironment within patients with aortic disease, suggesting a spectrum of bacterial communities that progress from blood through the luminal thrombus to the aortic wall. Further work could distinguish genus level variations that associates with disease in the different environments.