Multiple DNA Research Articles

Mutations to transcription factor MAX allosterically increase DNA selectivity by altering folding and binding pathways

Understanding how proteins discriminate between preferred and non-preferred ligands (‘selectivity’) is essential for predicting biological function and a central goal of protein engineering efforts, yet the biophysical mechanisms underpinning selectivity remain poorly understood. Towards this end, we study how variants of the promiscuous transcription factor (TF) MAX (H. sapiens) alter DNA specificity and selectivity, yielding >1700 Kds and >500 rate constants in complex with multiple DNA sequences. Twenty-two of the 240 assayed MAX point mutations enhance selectivity, yet none of these mutations occur at residues that contact nucleotides in published structures. By applying thermodynamic and kinetic models to these results and previous observations for the highly similar yet far more selective TF Pho4 (S. cerevisiae), we find that these mutations enhance selectivity by altering partitioning between or affinity within conformations with different intrinsic selectivity, providing a mechanistic basis for allosteric modulation of ligand selectivity. These results highlight the importance of conformational heterogeneity in determining sequence selectivity and can guide future efforts to engineer selective proteins.

Misuse of the Lower Limit of Detection in HBV DNA Testing and Anti-HBe Positive Status Will Significantly Impact the Diagnosis of Occult HBV Infection.

The diagnosis of occult hepatitis B virus (HBV) infection (OBI) is influenced by factors such as the lower limit of detection (LOD) of the HBV DNA test. However, in clinical practice and scientific research, the lower limit of quantification (LOQ) is often misused as the LOD. This study aims to investigate the impact of misuse of the LOD of the HBV DNA test on the detection rate of OBI, as well as the risk factors for OBI. Four hundred twelve patients who were HBsAg-negative and had undergone high-sensitivity HBV DNA testing were included in this study. HBV DNA was detected using the Cobas 6800 System with an LOD of 2.4 IU/mL and an LOQ of 10 IU/mL. The effect of using the LOQ as the LOD on the detection rate of OBI was compared, and univariate and multivariate logistic regression analyses were used to explore the risk factors for OBI. (1) Of the 412 patients, 63.3% (n = 261) were male, with a median age of 47 (range 34-55) years. A total of 473 HBV DNA test results were obtained, with 366 individuals undergoing only one HBV DNA test and the remaining 46 patients undergoing 2 to 5 HBV DNA tests (resulting in a total of 107 test results). (2) Considering only the first HBV DNA test result, the detection rate of OBI was 4.1% (17/412). However, when the LOQ (10 IU/mL) was used as the LOD, the detection rate of OBI was only 1.5% (6/412) (p < 0.001). (3) Univariate analysis showed that there were statistically significant differences in age, anti-HBe positivity rate and anti-HBc positivity rate between OBI and non-OBI individuals (p < 0.05). Multivariate regression analysis showed that anti-HBe positivity was an independent risk factor for OBI in this study (odds ratio [OR] = 3.807, 95% confidence interval [CI]: 1.065-13.617, p = 0.040), while anti-HBs positivity was a protective factor against OBI (OR = 0.271, 95% CI: 0.093-0.787, p = 0.016). (4) Among the 46 patients who underwent repeated testing, a total of seven individuals were found to be HBV DNA-positive in the first test, and six individuals tested positive for HBV DNA one or more times in subsequent tests. When OBI was confirmed by ≥ 1 out of 1-5 tests with detectable HBV DNA, the detection rate of OBI in this study could increase from 4.1% to 5.6%. The detection rate of OBI among HBsAg-negative adult patients attending hepatology departments in this region is 4.1%. Misusing the LOQ as LOD can significantly decrease the detection rate of OBI. The presence of anti-HBe positivity and undergoing multiple HBV DNA tests can lead to a significant increase in the detection rate of OBI.

YeastFab Cloning of Toxic Genes and Protein Expression Optimization in Yeast.

YeastFab is a Golden Gate-based cloning standard and parts repository. It is designed for modular, hierarchical assembly of transcription units and multi-gene assemblies for expression in Saccharomyces cerevisiae. This makes it a suitable toolbox to optimize the expression strength of heterologous genes in yeast. When cloning heterologous coding sequences into YeastFab vectors, in several cases we have observed toxicity to the cloning host Escherichia coli. The provided protocol details how to clone such toxic genes from multiple synthetic DNA fragments while adhering to the YeastFab standard. The presented cloning strategy includes a C-terminal FLAG tag that allows screening for constructs with a desired protein expression in yeast by western blot. The design allows scarlessly removing the tag through a Golden Gate reaction to facilitate cloning of expression constructs with the native, untagged transgene.

Using ApE for In Silico Golden Gate Cloning.

Golden Gate cloning allows rapid and reliable assembly of multiple DNA fragments in a defined orientation. Golden Gate cloning requires careful design of the restriction fragment overhangs to minimize undesired products and to generate the desired junctions. The ApE (A plasmid Editor) software package can assist in silico design of input fragments or to generate expected assembly products.

Prognostic and immunological role of LASP2 in clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) represents a common renal carcinoma subtype influenced by the immune microenvironment. LIM and SH3 Protein 2 (LASP2), an actin-binding protein within the nebulin family, contributes to cellular immunity and adhesion mechanisms. This study aimed to clarify the immunological and prognostic relevance of LASP2 in ccRCC. Using clinical and expression data from TCGA, LASP2 expression levels were analyzed alongside clinicopathological features in ccRCC patients. Validation was conducted through real-world samples and tissue microarrays. Comprehensive analysis using online databases examined genetic mutations, DNA methylation patterns, and immune microenvironment characteristics. Gene set enrichment analysis (GSEA) provided insights into LASP2's potential mechanisms in ccRCC. LASP2 expression was notably reduced and correlated with adverse clinicopathological features and prognosis in ccRCC patients. Prognostic associations were identified across multiple CpG DNA methylation sites. LASP2 levels showed significant correlations with immune cell infiltration and checkpoint genes, including PDCD1 and CTLA4. GSEA findings highlighted LASP2's enrichment within metabolic pathways and signaling networks, such as fatty acid metabolism, TGF-β signaling, and epithelial-mesenchymal transition. LASP2 emerged as an immune-associated biomarker linked to poorer survival outcomes in ccRCC, suggesting its potential as a novel anti-cancer target and prognostic indicator in ccRCC.

Multiple DNA repair pathways prevent acetaldehyde-induced mutagenesis in yeast.

Acetaldehyde is the primary metabolite of alcohol and is present in many environmental sources including tobacco smoke. Acetaldehyde is genotoxic, whereby it can form DNA adducts and lead to mutagenesis. Individuals with defects in acetaldehyde clearance pathways have increased susceptibility to alcohol-associated cancers. Moreover, a mutation signature specific to acetaldehyde exposure is widespread in alcohol and smoking-associated cancers. However, the pathways that repair acetaldehyde-induced DNA damage and thus prevent mutagenesis are vaguely understood. Here, we used Saccharomyces cerevisiae to delete genes in each of the major DNA repair pathways to identify those that alter acetaldehyde-induced mutagenesis. We observed that loss of functional nucleotide excision repair (NER) had the largest effect on acetaldehyde mutagenesis. In addition, base excision repair (BER), as well as DNA protein crosslink (DPC) repair pathways were involved in modulating acetaldehyde mutagenesis, while mismatch repair (MMR), homologous recombination (HR) and post replication repair are dispensable for acetaldehyde mutagenesis. Acetaldehyde-induced mutations in an NER-deficient (Δrad1) background were dependent on translesion synthesis as well as DNA inter-strand crosslink (ICL) repair. Moreover, whole genome sequencing of the mutated isolates demonstrated an increase in C→A changes coupled with an enrichment of gCn→A changes which is diagnostic of acetaldehyde exposure in yeast and in human cancers. Finally, downregulation of the leading strand replicative polymerase Pol epsilon, but not the lagging strand polymerase, resulted in increased acetaldehyde mutagenesis, indicating that lesions are likely formed on the leading strand. Our findings demonstrate that multiple DNA repair pathways coordinate to prevent acetaldehyde-induced mutagenesis.

The effect of modifications to DNA structure on the architecture of bacterial DNA condensed with a nucleoid-associated protein

Bacteria are adaptive organisms that have developed various protective mechanisms to respond to a range of environmental stressors like nutrient deprivation. One distinct protective mechanism in Escherichia coli that will be discussed further involves the overproduction of a nucleoid-associated protein called DNA-binding protein (Dps) from starved cells. The high concentration of Dps protects the bacteria’s genetic material by binding to the DNA and forming a Dps-DNA condensate. The morphology of these Dps-DNA condensates in an in-vitro system will change under different environmental conditions. To better understand how altering Dps-DNA interactions independent of environmental stressors affects the resulting morphology of the Dps-DNA condensate in an in-vitro system, fluorescence microscopy imaging was utilized to examine the characteristics of the condensate in response to different DNA features. Upon varying the length of linear DNA fragments incubated with Dps protein, the lengths of the DNA did not change the morphology/structure of the condensate. However, incubation with linear 5k DNA fragments resulted in a greater amount for and larger sized condensate formation. When Dps was incubated with DNA fragments that were either supercoiled or relaxed, minimal differences were observed in the size and structure of condensates. These findings demonstrate that Dps can bind to and condense multiple DNA conformations, helping better explain the function of Dps in organisms other than E. coli.

Accurate phenotype-to-genotype mapping of high-diversity yeast libraries by heat-shock-electroporation (HEEL).

High-throughput DNA transformation techniques are invaluable when generating high-diversity mutant libraries, a cornerstone of successful protein engineering. However, transformation efficiencies have a direct correlation with the probability of introducing multiple DNA molecules into each cell, although reliable library screenings require cells that contain a single unique genotype. Thus, transformation methods that yield a high multiplicity of transformations are unsuitable for high-diversity library screenings. Here, we describe an innovative yeast library transformation method that is both simple and highly efficient. Our dual heat-shock and electroporation approach (HEEL) creates high-quality DNA libraries by increasing the fraction of mono-transformed yeast cells from 20% to over 70% of all transformed cells, thus allowing for near-perfect phenotype-to-genotype associations. HEEL also allows more than 107 yeast cells per reaction to be transformed with a circular plasmid molecule, which corresponds to an almost 100-fold improvement compared with current yeast transformation methods. To further refine our library screening approach, we integrated an automated yeast genotyping workflow with a dual-barcode design that employs both a single nucleotide polymorphism and a high-diversity region. This design allows for robust identification and quantification of unique genotypes within a heterogeneous population using standard Sanger sequencing. Our findings demonstrate that the longstanding trade-off between the size and quality of transformed yeast libraries can be overcome. By employing the HEEL method, large DNA libraries can be transformed into yeast with high-efficiency, while maintaining high library quality, essential for successful mutant screenings. This advancement holds significant promise for the fields of molecular biology and protein engineering.IMPORTANCEWith the recent expansion of artificial intelligence in the field of synthetic biology, there has never been a greater need for high-quality data and reliable measurements of phenotype-to-genotype relationships. However, one major obstacle to creating accurate computer-based models is the current abundance of low-quality phenotypic measurements originating from numerous high-throughput but low-resolution assays. Rather than increasing the quantity of measurements, new studies should aim to generate as accurate measurements as possible. The HEEL methodology presented here aims to address this issue by minimizing the problem of multi-plasmid uptake during high-throughput yeast DNA transformations, which leads to the creation of heterogeneous cellular genotypes. HEEL should enable highly accurate phenotype-to-genotype measurements going forward, which could be used to construct better computer-based models.

GDF-15 as a proxy for epigenetic aging: associations with biological age markers, and physical function

Growth differentiation factor 15 (GDF-15) has emerged as a significant biomarker of aging, linked to various physiological and pathological processes. This study investigates circulating GDF-15 levels in a cohort of healthy individuals from the Balearic Islands, exploring its associations with biological age markers, including multiple DNA methylation (DNAm) clocks, physical performance, and other age-related biomarkers. Seventy-two participants were assessed for general health, body composition, and physical function, with GDF-15 levels quantified using ELISA. Our results indicate that GDF-15 levels significantly increase with age, particularly in individuals over 60. Strong positive correlations were observed between GDF-15 levels and DNAm GrimAge, DNAm PhenoAge, Hannum, and Zhang clocks, suggesting that GDF-15 could serve as a proxy for epigenetic aging. Additionally, GDF-15 levels were linked to markers of impaired glycemic control, systemic inflammation, and physical decline, including decreased lung function and grip strength, especially in men. These findings highlight the use of GDF-15 as a biomarker for aging and age-related functional decline. Given that GDF-15 is easier to measure than DNA methylation, it has the potential to be more readily implemented in clinical settings for broader health assessment and management.

Microchimerism: The mystery of multiple DNA and its implications in forensic sciences.

Microchimerism (MC) refers to the presence of small amounts of foreign cells or DNA in the tissues or circulation of an individual. It generally occurs through mother-fetus interaction, twin pregnancies, and intergenerational transmission. MC is influenced by genetic and environmental factors such as toxic conditions, immunological suppression, and various diseases (influenza, COVID-19, etc.). Progenitor cells transferred from the fetus to the mother through fetal MC are known to differentiate into neurons in the maternal brain. Although the relationship between these cells and the brain is not fully understood, it is thought that they may play a role in the emergence of some mental illnesses. The long-term presence of microchimeric cells in the body by differentiating into various cell types such as the brain, heart, bone, liver, and lung can lead to the presence of two or more DNA sets in an individual. This can lead to confusion in forensic identification and sex determination processes. This review aims to provide a comprehensive review of the definition, transmission pathways, detection duration in the human body, associated diseases, analytical detection techniques, and the importance of MC in forensic sciences. In this context, it is aimed to draw attention to the potential dangers of MC and contribute to the justice system. Furthermore, this study emphasizes the need for scientific research on this topic by creating a starting point for future research in the field of MC.

Sanguinarine identified as a natural dual inhibitor of AURKA and CDK2 through network pharmacology and bioinformatics approaches

Cervical cancer (CA) continues to be a female malignant tumor with limited therapeutic options, resulting in a high mortality rate. Sanguinarine (SANG), a naturally occurring alkaloid, has demonstrated notable efficacy in preclinical treatment of CA. However, the mechanism through which SANG acts against CA is not fully understood. To address this, utilizing nine drug target prediction databases, we have successfully identified 379 potential targets for SANG. Venn diagram analysis compared 2367 CA-related targets from the GeneCards disease database, 2618 CA-closely related targets derived from multiple datasets in GEO through WGCNA analysis, and the 379 potential targets of SANG, resulting in 35 shared targets. Subsequently, by employing PPI network analysis, the Cytohubba plugin, the Human Protein Atlas, TCGA database data, and ROC curve analysis, we have identified AURKA and CDK2 as key targets of SANG in combating CA. Single-gene GSEA results suggest that the overexpression of AURKA and CDK2 is closely correlated with DNA replication, cell cycle progression, and various DNA repair pathways in CA. Molecular docking and molecular simulation dynamics analyses have confirmed the stable binding of both AURKA and CDK2 to SANG. In summary, by integrating diverse methodological approaches, this study discovered that SANG potentially inhibits the malignant features of CA by targeting AURKA and CDK2, thereby regulating DNA replication, cell cycle progression, and multiple DNA repair pathways. This lays a solid foundation for further exploring the pharmacological role of SANG in CA therapy. However, further in-depth in vitro and in vivo experiments are required to corroborate our findings.

Bioinspired designer DNA NanoGripper for virus sensing and potential inhibition.

DNA has shown great biocompatibility, programmable mechanical properties, and precise structural addressability at the nanometer scale, rendering it a material for constructing versatile nanorobots for biomedical applications. Here, we present the design principle, synthesis, and characterization of a DNA nanorobotic hand, called DNA NanoGripper, that contains a palm and four bendable fingers as inspired by naturally evolved human hands, bird claws, and bacteriophages. Each NanoGripper finger consists of three phalanges connected by three rotatable joints that are bendable in response to the binding of other entities. NanoGripper functions are enabled and driven by the interactions between moieties attached to the fingers and their binding partners. We demonstrate that the NanoGripper can be engineered to effectively interact with and capture nanometer-scale objects, including gold nanoparticles, gold NanoUrchins, and SARS-CoV-2 virions. With multiple DNA aptamer nanoswitches programmed to generate a fluorescent signal that is enhanced on a photonic crystal platform, the NanoGripper functions as a highly sensitive biosensor that selectively detects intact SARS-CoV-2 virions in human saliva with a limit of detection of ~100 copies per milliliter, providing a sensitivity equal to that of reverse transcription quantitative polymerase chain reaction (RT-qPCR). Quantified by flow cytometry assays, we demonstrated that the NanoGripper-aptamer complex can effectively block viral entry into the host cells, suggesting its potential for inhibiting virus infections. The design, synthesis, and characterization of a sophisticated nanomachine that can be tailored for specific applications highlight a promising pathway toward feasible and efficient solutions to the detection and potential inhibition of virus infections.

Multiple Displacement Amplification Facilitates SMRT Sequencing of Microscopic Animals and the Genome of the Gastrotrich Lepidodermella squamata (Dujardin 1841).

Obtaining adequate DNA for long-read genome sequencing remains a roadblock to producing contiguous genomes from small-bodied organisms, hindering understanding of phylogenetic relationships and genome evolution. Multiple displacement amplification leverages Phi29 DNA polymerase to produce micrograms of DNA from picograms of input. However, multiple displacement amplification's inherent biases in amplification related to guanine and cytosine (GC) content, repeat content and chimera production are a problem for long-read genome assembly, which has been little investigated. We explored the utility of multiple displacement amplification for generating template DNA for High Fidelity (HiFi) sequencing directly from living cells of Caenorhabditis elegans (Nematoda) and Lepidodermella squamata (Gastrotricha) containing one order of magnitude less DNA than required for the PacBio Ultra-Low DNA Input Workflow. High Fidelity sequencing of libraries prepared from multiple displacement amplification products resulted in highly contiguous and complete genomes for both C. elegans (102 Mbp assembly; 336 contigs; N50 = 868 kbp; L50 = 39; BUSCO_nematoda_nucleotide: S:96.1%, D:2.8%) and L. squamata (122 Mbp assembly; 157 contigs; N50 = 3.9 Mbp; L50 = 13; BUSCO_metazoa_nucleotide: S:80.8%, D:2.8%). Coverage uniformity for reads from multiple displacement amplification DNA (Gini Index: 0.14, normalized mean across all 100 kbp blocks: 0.49) and reads from pooled nematode DNA (Gini Index: 0.16, normalized mean across all 100 kbp blocks: 0.49) proved similar. Using this approach, we sequenced the genome of the microscopic invertebrate L. squamata (Gastrotricha), the first of its phylum. Using the newly sequenced genome, we infer Gastrotricha's long-debated phylogenetic position as the sister taxon of Platyhelminthes and conduct a comparative analysis of the Hox cluster.

Converting Multiple- to Single-DNA-Tethered Beads and Removing Only-One-End-Tethered DNA in High-Throughput Stretching.

S-DNA is a double-stranded DNA that forms under tensions of >65 pN. Here, we report that S-DNA resists the cleavage of Cas12a and the restriction endonuclease SmaI. Taking advantage of this resistance, in magnetic tweezer experiments, we developed an assay to convert multiple-DNA-tethered beads into single-DNA-tethered beads and remove the only-one-end-tethered DNA molecule by cleaving the DNA that does not transition to S-DNA at about 80 pN. When multiple DNA molecules are tethered to a single bead, they share the tension, exist in the B-form, and allow the cleavage. Only-one-end-tethered DNA molecules, free of tension, are also cleaved. In versatile types of experiments, we proved the broad applications of this assay: measuring the correct DNA elasticity and DNA condensation dynamics by avoiding the false results due to interference of only-one-end-tethered DNA molecules and quantifying the accurate cleavage rates of Cas12a and the restriction endonucleases by eliminating the error caused by multiple-DNA-tethered beads. This convenient assay ensures correct and accurate results in high-throughput DNA stretching experiments.

Horizontal transfer of plasmid-like extrachromosomal circular DNAs across graft junctions in Solanaceae

The transfer of genetic material between stocks and scions of grafted plants has been extensively studied; however, the nature and frequency of the transferred material remain elusive. Here, we report a grafting system involving woody goji as the stock and herbaceous tomato as the scion, which was developed using in vitro and in vivo approaches; the results confirmed horizontal transfer of multiple nuclear DNA fragments from donor goji cells to recipient tomato cells. Tomato tissues containing goji donor DNA fragments at or near the grafting junctions had a perennial-biased anatomical structure, from which roots or shoots were regenerated. Most of the fragments were plasmid-like extrachromosomal circular DNAs (eccDNAs) present in the regenerants derived from the cells and in their asexual offspring. Plants with transferred eccDNAs in regenerated roots or shoots (designated “Go-tomato”) were grown perennially and showed excellent agronomic performance. The present study provides new insights into the replication, expression, and potential function of eccDNAs in the pleiotropic traits of Go-tomato. Mobile eccDNAs offer evidence of stock-to-scion horizontal DNA transfer beyond chromosomes and organelles, thereby contributing to the molecular understanding of graft-induced genetic variation, evolution, and breeding.

A multiple signal amplification electrochemiluminescence sensor for Hg2+ detection based on Ce2Sn2O7/poly(5-formylindole) nanocomposites

A novel electrochemiluminescence (ECL) sensor for the detection of Hg2+ is constructed based on the synthesized cerium stannate/poly(5-formylindole) (Ce2Sn2O7/P5FIn) composite and multiple DNA signal amplification techniques, including strand displacement amplification (SDA) reaction and hybridization chain reaction (HCR). On the one hand, the combination of Ce2Sn2O7 and P5FIn improves the electrochemical reaction rate between Ce2Sn2O7 and the co-reactant, and significantly enhances the ECL signal. On the other hand, compared to P5FIn, Ce2Sn2O7/P5FIn has a larger specific surface area to facilitate the effective fixation of hairpin DNA H1. The target Hg2+ is bound to the DNA strand (mDNA) rich in thymine bases (T) by T-Hg2+-T. Subsequently, with the assistance of polymerase (phi29 DNA) and restriction endonuclease (Nt.BbvCI), strand displacement amplification (SDA) reaction is triggered to generate a large number of simulated target (MT). Hairpin DNA H1 captures MT, which then further triggers a hybridization chain reaction (HCR) to produce long strand DNA (dsDNA) rich in cytosine. Finally, a large amount of Ag+ is introduced through C–Ag+-C, which is used as a co-reaction accelerator of Ce2Sn2O7–K2S2O8 system to significantly enhance the ECL signal strength. Under optimal conditions, the constructed ECL sensor can detect Hg2+ in a wide linear response range of 1.0 fM-100 nM, with a detection limit of 0.30 fM. The sensor also has high sensitivity and good applicability in the detection of seawater samples.

Novel analysis tool for the distance of gold dimers controlled by the DNA strand length on the DNA origami.

Metallic nanoparticle dimers have been used to enhance the excitation rate of single-quantum emitters. The interparticle distance (d) of the dimers has a crucial influence on the signal enhancement. Therefore, precise control of d is desired for optimal performance. However, statistical analysis of d has been often restricted to a small number of dimers due to the lack of reliable automatic software tools. For this reason, we developed a novel analysis tool for automatic dimer analysis. Our approach combines particle detection by circle Hough transformation (CHT) with custom classification routines optimised for distinct types of particles. We applied our tool to scanning electron microscopy (SEM) images and achieved great agreement in dimer detection, reaching an agreement of around 97% between automatic analysis and manual inspection for more than 3000 metallic nanoparticle dimers on DNA origami controlled by a combination of multiple DNA strands. Our study revealed the effects of the strand length (L) on the distribution of d. Based on geometric consideration, we expected a strong correlation between L and the standard deviation (σ) of d. We could verify this correlation by characterising four dimer designs with different L while analysing more than 1000 dimers per specimen.

Highly specific multiplex DNA methylation detection for liquid biopsy of colorectal cancer

BackgroundCirculating tumor DNA (ctDNA) has emerged as a useful biomarker for cancer detection and prognosis. In this study, we developed a strategy for developing a highly specific multiplex qPCR assay to detect methylated ctDNA in the blood of colorectal cancer (CRC) patients and investigated the potential use for the detection and prognosis of CRC. MethodsBisulfite conversion and amplicon sequencing were used to confirm potential CRC-specific DNA methylation markers. The selected DNA methylation candidates were validated by qMSP. The six best-performing markers were used to develop a new single-tube multiplex quantitative methylation-specific PCR assay (mqMSP). The mqMSP assay was applied to analyze plasma samples from 114 CRC patients, 47 patients with advanced adenoma, 45 patients with benign polyps, and 57 healthy controls. The clinical performance of the assay and associations with clinical outcomes were assessed. ResultsSix DNA methylation biomarkers were confirmed to be specifically hypermethylated in CRC tumor tissues. The newly developed mqMSP assay detected CRC with extremely high specificity (specificity of 98.2 %, with sensitivity of 67.5 %). The detection rate of ctDNA was significantly correlated with tumor size and clinical stage, with ctDNA methylation levels in the blood markedly increased with larger tumor size, poor differentiation, and advanced stage. Moreover, high preoperative methylated ctDNA level was associated with worse recurrence-free survival and overall survival. ConclusionWe provided a strategy for identification of multiple highly-specific DNA methylation markers for designing multiplex DNA methylation assays for liquid biopsies of CRC. The newly developed assay has potential for CRC early detection, and prognosis.

Reference Sequence Browser: An R application with a user-friendly GUI to rapidly query sequence databases.

Land managers, researchers, and regulators increasingly utilize environmental DNA (eDNA) techniques to monitor species richness, presence, and absence. In order to properly develop a biological assay for eDNA metabarcoding or quantitative PCR, scientists must be able to find not only reference sequences (previously identified sequences in a genomics database) that match their target taxa but also reference sequences that match non-target taxa. Determining which taxa have publicly available sequences in a time-efficient and accurate manner currently requires computational skills to search, manipulate, and parse multiple unconnected DNA sequence databases. Our team iteratively designed a Graphic User Interface (GUI) Shiny application called the Reference Sequence Browser (RSB) that provides users efficient and intuitive access to multiple genetic databases regardless of computer programming expertise. The application returns the number of publicly accessible barcode markers per organism in the NCBI Nucleotide, BOLD, or CALeDNA CRUX Metabarcoding Reference Databases. Depending on the database, we offer various search filters such as min and max sequence length or country of origin. Users can then download the FASTA/GenBank files from the RSB web tool, view statistics about the data, and explore results to determine details about the availability or absence of reference sequences.

Multiple-resistance evolution to ACCase inhibitors and glyphosate in sourgrass (Digitaria insularis) is attributed to diverse polymorphisms in the herbicide target sites

Abstract Sourgrass [Digitaria insularis (L.) Mez ex Ekman] is considered the most troublesome weed in agronomic crops in South America. Overreliance on glyphosate has selected for resistant populations, although the resistance mechanisms remain unknown. Recently, populations were identified that exhibited multiple resistance to 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) and acetyl-CoA carboxylase (ACCase) inhibitors, posing a significant challenge due to the lack of alternative control options. This project aimed to identify the resistance patterns and levels to glyphosate and ACCase inhibitors of three suspected resistant populations (P1, P2, and P3), and elucidate the resistance mechanisms. We performed dose–response experiments with clethodim, fluazifop-P-butyl, glyphosate, and pinoxaden to identify the possibility of cross- and multiple resistance and to quantify the resistance levels. We sequenced the ACCase and EPSPS genes to test the hypothesis that target-site mutations were involved in the resistance mechanisms, given the resistance patterns observed. Our results indicated that two of the tested populations, P1 and P2, were multiple resistant to glyphosate and all ACCase-inhibitor classes, while P3 was resistant to glyphosate only. Resistance levels varied by herbicide, with resistance indices ranging from 2.7- to nearly 2,000-fold. We identified an amino acid substitution in ACCase at position 2078 (Asp-2078-Gly), homozygous for both P1 and P2, corroborating the resistance patterns observed. Interestingly, EPSPS sequencing identified multiple heterozygous DNA polymorphisms that resulted in amino acid substitutions at positions 106 (P1 and P2) or at both 102 and 106 (P3), indicating multiple evolutionary origins of glyphosate-resistance evolution. We show for the first time the genetic mechanisms of multiple resistance to glyphosate and ACCase in D. insularis, and provide a thorough discussion of the evolutionary and management implications of our work.