DNA Degradation Research Articles

CRISPR-Cas12a based target recognition initiated duplex-specific nuclease enhanced fluorescence and colorimetric analysis of cell-free DNA (cfDNA)

The significant role of cell-free DNA (cfDNA) for disease diagnosis, including cancer, has garnered a lot of attention. The challenges of creating target-specific primers and the possibility of false-positive signals make amplification-based detection methods problematic. Fluorescent biosensors based on CRISPR-Cas have been widely established, however they still require an amplification step before they can be used for detection. To detect cfDNA, researchers have created a CRISPR-Cas12a-based nucleic acid amplification-free fluorescent biosensor that uses a combination of fluorescence and colorimetric signaling improved by duplex-specific nuclease (DSN). DSN-assisted signal recycling is initiated in H1@MBs when the target cfDNA activates the CRISPR-Cas12a complex, leading to the degradation of single-strand DNA (ssDNA) sequences. This method has an extremely high detection limit for the BRCA-1 breast cancer gene. In addition to measuring viral DNA in a field-deployable and point-of-care testing (POCT) platform, this fast and highly selective sensor can be used to evaluate additional nucleic acid biomarkers.

The Influence of Tanning Chemical Agents on DNA Degradation: A Robust Procedure for the Analysis of Tanned Animal Hide-A Pilot Study.

Illegal wildlife trade is currently on the rise, and it is becoming one of the most lucrative crime sectors. The rarer the species, the higher the demand. Wildlife trade falls under international regulations, such as the CITES convention. Proving that this convention has been violated is a complex process and can be very difficult to do. DNA analysis methods remain (in many cases) the only way to determine whether a certain specimen originated from a protected animal species, a specific individual, or a species in which it is legal to trade. Tanned animal hides are a specific type of specimen. With this type of biological material, obtaining amplifiable DNA is often difficult. This pilot study aimed to map the effect of the chemicals used in the tanning process on the degradation of the DNA yielded from such samples. The DNA was quantified using two different approaches: qPCR and Qubit fluorometry. The degree of DNA fragmentation was assessed by determining the degradation index. The results indicate that reagents containing chromium have the greatest influence on DNA degradation. However, by using the presented protocol, enough amplifiable DNA can be obtained from hides treated with aluminum-based reagents.

Kaposi's sarcoma-associated herpesvirus (KSHV) LANA prevents KSHV episomes from degradation.

Protein knockdown with an inducible degradation system is a powerful tool for studying proteins of interest in living cells. Here, we adopted the auxin-inducible degron (AID) approach to detail Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) function in latency maintenance and inducible viral lytic gene expression. We fused the mini-auxin-inducible degron (mAID) tag at the LANA N-terminus with KSHV bacterial artificial chromosome 16 recombination, and iSLK cells were stably infected with the recombinant KSHV encoding mAID-LANA. Incubation with 5-phenyl-indole-3-acetic acid, a derivative of natural auxin, rapidly degraded LANA within 1.5 h. In contrast to our hypothesis, depletion of LANA alone did not trigger lytic reactivation but rather decreased inducible lytic gene expression when we stimulated reactivation with a combination of ORF50 protein expression and sodium butyrate. Decreased overall lytic gene induction seemed to be associated with a rapid loss of KSHV genomes in the absence of LANA. The rapid loss of viral genomic DNA was blocked by a lysosomal inhibitor, chloroquine. Furthermore, siRNA-mediated knockdown of cellular innate immune proteins, cyclic AMP-GMP synthase (cGAS) and simulator of interferon genes (STING), and other autophagy-related genes rescued the degradation of viral genomic DNA upon LANA depletion. Reduction of the viral genome was not observed in 293FT cells that lack the expression of cGAS. These results suggest that LANA actively prevents viral genomic DNA from sensing by cGAS-STING signaling axis, adding novel insights into the role of LANA in latent genome maintenance.IMPORTANCESensing of pathogens' components is a fundamental cellular immune response. Pathogens have therefore evolved strategies to evade such cellular immune responses. KSHV LANA is a multifunctional protein and plays an essential role in maintaining the latent infection by tethering viral genomic DNA to the host chromosome. We adopted the inducible protein knockdown approach and found that depletion of LANA induced rapid degradation of viral genomic DNA, which is mediated by innate immune DNA sensors and autophagy pathway. These observations suggest that LANA may play a role in hiding KSHV episome from innate immune DNA sensors. Our study thus provides new insights into the role of LANA in latency maintenance.

Wide-spread dispersal in a deep-sea brooding polychaete: the role of natural history collections in assessing the distribution in quill worms (Onuphidae, Annelida)

BackgroundModern integrative taxonomy-based annelid species descriptions are detailed combining morphological data and, since the last decades, also molecular information. Historic species descriptions are often comparatively brief lacking such detail. Adoptions of species names from western literature in the past led to the assumption of cosmopolitan ranges for many species, which, in many cases, were later found to include cryptic or pseudocryptic lineages with subtle morphological differences. Natural history collections and databases can aid in assessing the geographic ranges of species but depend on correct species identification. Obtaining DNA sequence information from wet-collection museum specimens of marine annelids is often impeded by the use of formaldehyde and/or long-term storage in ethanol resulting in DNA degradation and cross-linking.ResultsThe application of ancient DNA extraction methodology in combination with single-stranded DNA library preparation and target gene capture resulted in successful sequencing of a 110-year-old collection specimen of quill worms. Furthermore, a 40-year-old specimen of quill worms was successfully sequenced using a standard extraction protocol for modern samples, PCR and Sanger sequencing. Our study presents the first molecular analysis of Hyalinoecia species including the previously known species Hyalinoecia robusta, H. tubicloa, H. artifex, and H. longibranchiata, and a potentially undescribed species from equatorial western Africa morphologically indistinguishable from H. tubicola. The study also investigates the distribution of these five Hyalinoecia species. Reassessing the distribution of H. robusta reveals a geographical range covering both the Atlantic and the Indian Oceans as indicated by molecular data obtained from recent and historical specimens.ConclusionOur results represent an example of a very wide geographical distribution of a brooding deep-sea annelid with a complex reproduction strategy and seemingly very limited dispersal capacity of its offspring, and highlights the importance of molecular information from museum specimens for integrative annelid taxonomy and biogeography.

Evaluation of ancient DNA imputation: a simulation study

Ancient genomic data is becoming increasingly available thanks to recent advances in high-throughput sequencing technologies. Yet, post-mortem degradation of endogenous ancient DNA often results in low depth of coverage and subsequently high levels of genotype missingness and uncertainty. Genotype imputation is a potential strategy for increasing the information available in ancient DNA samples and thus improving the power of downstream population genetic analyses. However, the performance of genotype imputation on ancient genomes under different conditions has not yet been fully explored, with all previous work primarily using an empirical approach of downsampling high coverage paleogenomes. While these studies have provided invaluable insights into best practices for imputation, they rely on a fairly limited number of existing high coverage samples with significant temporal and geographical biases. As an alternative, we used a coalescent simulation approach to generate genomes with characteristics of ancient DNA in order to more systematically evaluate the performance of two popular imputation software, BEAGLE and GLIMPSE, under variable divergence times between the target sample and reference haplotypes, as well as different depths of coverage and reference sample size. Our results suggest that for genomes with coverage <=0.1x imputation performance is poor regardless of the strategy employed. Beyond 0.1x coverage imputation is generally improved as the size of the reference panel increases, and imputation accuracy decreases with increasing divergence between target and reference populations. It may thus be preferable to compile a smaller set of less diverged reference samples than a larger more highly diverged dataset. In addition, the imputation accuracy may plateau beyond some level of divergence between the reference and target populations. While accuracy at common variants is similar regardless of divergence time, rarer variants are better imputed on less diverged target samples. Furthermore, both imputation software, but particularly GLIMPSE, overestimate high genotype probability calls, especially at low coverages. Our results provide insight into optimal strategies for ancient genotype imputation under a wide set of scenarios, complementing previous empirical studies based on imputing downsampled high-coverage ancient genomes.

Antiproliferative activity of platinum(II) and copper(II) complexes containing novel biquinoxaline ligands.

Nowadays, cancer represents one of the major causes of death in humans worldwide, which renders the quest for new and improved antineoplastic agents to become an urgent issue in the field of biomedicine and human health. The present research focuses on the synthesis of 2,3,2',3'-tetra(pyridin-2-yl)-6,6'-biquinoxaline) and (2,3,2',3'-tetra(thiophen-2-yl)-6,6'-biquinoxaline) containing copper(II) and platinum(II) compounds as prodrug candidates. The binding interaction of these compounds with calf thymus DNA (CT-DNA) and human serum albumin were assessed with UV titration, thermal decomposition, viscometric, and fluorometric methods. The thermodynamical parameters and the temperature-dependent binding constant (K'b) values point out to spontaneous interactions between the complexes and CT-DNA via the van der Waals interactions and/or hydrogen bonding, except Cu(ttbq)Cl2 for which electrostatic interaction was proposed. The antitumor activity of the complexes against several human glioblastomata, lung, breast, cervix, and prostate cell lines were investigated by examining cell viability, oxidative stress, apoptosis-terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, in vitro migration and invasion, in vitro-comet DNA damage, and plasmid DNA interaction assays. The U87 and HeLa cells were investigated as the cancer cells most sensitive to our complexes. The exerted cytotoxic effect of complexes was attributed to the formation of the reactive oxygen species in vitro. It is clearly demonstrated that Cu(ttbq)Cl2, Pt(ttbq)Cl2, and Pt(tpbq)Cl2 have the highest DNA degradation potential and anticancer effect among the tested complexes by leading apoptosis. The wound healing and invasion analysis results also supported the higher anticancer activity of these two compounds.

DNA degradation of bloodstains on cotton fabric caused by different washing procedures.

DNA degradation in biological material needs to be better understood. Bloodstains on washed clothing are disturbed by washing procedures, sometimes transferred to other fabrics, often with latent bloodstains and usually with significantly degraded DNA.The samples (cotton fabric with bloodstains) are divided into six main groups, depending on the washing method regarding water temperature (95, 60, and 30°C) and the detergent use. After completing the washing process, samples were stored for a certain period (1day to 6months) and subsequently analyzed. Analyses were performed using standard protocols and commercial kits to measure the remaining DNA quantity (concentration) and DNA degradation index in the processed samples.Our results revealed that the high washing temperature (60 and 95°C) and the application of detergent have a synergic action on DNA degradation, while at 30°C this effect is absent. Furthermore, the effect of detergent on accelerated DNA degradation is observed about a month after the washing. This delayed effect of detergent has no explanation in current literature data.To obtain optimal results from the bloodstains, we recommended that the period from the crime event and attempted cleaning by a perpetrator to the laboratory analysis should be less than 1month.

DNA degradation is involved with low physiological potential of soybean seeds

DNA degradation is involved with low physiological potential of soybean seeds

Role of DNase I in DNA degradation and cell-free DNA generation after acetaminophen-induced hepatic injury.

Cell-free DNA (cfDNA), the DNA in the blood circulation, is a useful marker for diagnosing hereditary diseases and tumors. However, the mechanisms underlying the generation of cfDNA are not completely understood. We previously studied DNases [Caspase-activated DNase (CAD), DNase1L3, and DNase I] and reported that in acetaminophen-induced liver necrosis, DNase1L3 was the main endonuclease generating cfDNA, with CAD playing a supporting role. In this study, we generated triple-gene knockout (TKO) mice, Cad -/-DNase1L3 -/-DNase1 -/-, and found that DNase I also contributed to cfDNA generation. Given that a defect in DNase1L3 or DNase I is involved in autoimmune diseases, TKO mice would be useful as a disease model and tool for identifying the in vivo roles of endonucleases.

Molecular identification of animal species of Bufonis Venenum from secretions

The animal species is one of the key factors affecting the quality of Bufonis Venenum. The quality of Bufonis Venenum derived from Bufo bufo gargarizans is significantly higher than that from B. melanostictus. Since Bufonis Venenum is from secretions, the conventional identification methods are difficult to identify the animal species due to the lack of the appearance and morphology of the animals. The rapid development of molecular identification technology has provided new methods for the identification of Bufonis Venenum. However, because of the low content and serve degradation of residual DNA in secretions, the research on the molecular identification of Chinese medicinal materials from secretions remains to be carried out. To understand the animal species of Bufonis Venenum, this study collected 83 samples of Bufonis Venenum, including 7 commercially available samples, 5 reference medicinal materials, and 71 animal samples from which Bufonis Venenum was prepared according to the method in the 2020 edition of the Chinese Pharmacopoeia. Different DNA extraction methods were used and compared, and the mitochondrial 16S rRNA gene fragments were amplified, on the basis of which the phylogenetic trees were built. Finally, molecular identification of the animal species of the samples was performed. The results showed that the DNA extracted from Bufonis Venenum by the reagent kit had good quality, and 16S rRNA sequences were successfully amplified from 80 out of the 83 samples. In addition, 71 16S rRNA sequences of the animal species of Bufonis Venenum were downloaded from GenBank. The phylogenetic trees constructed based on the neighbor-joining(NJ) method and the Bayesian inference(BI) method showed that the samples derived from B. bufo gargarizans and B. melanostictus were clustered into separate monophyletic clades, with the support of 100%(NJ) and 1.00(BI), respectively. The animal species of both commercially available samples and reference medicinal materials were B. bufo gargarizans. In conclusion, DNA can be extracted from Bufonis Venenum derived from secretions, and the 16S rRNA gene sequences can be amplified, which can be used for molecular identification of the animal species of Bufonis Venenum. The findings provide a reference for the quality control of Bufonis Venenum and the identification of animal species of medicinal materials derived from secretions.

STING inhibition enables efficient plasmid-based gene expression in primary vascular cells: A simple and cost-effective transfection protocol.

Plasmid transfection in cells is widely employed to express exogenous proteins, offering valuable mechanistic insight into their function(s). However, plasmid transfection efficiency in primary vascular endothelial cells (ECs) and smooth muscle cells (SMCs) is restricted with lipid-based transfection reagents such as Lipofectamine. The STING pathway, activated by foreign DNA in the cytosol, prevents foreign gene expression and induces DNA degradation. To address this, we explored the potential of STING inhibitors on the impact of plasmid expression in primary ECs and SMCs. Primary human aortic endothelial cells (HAECs) were transfected with a bicistronic plasmid expressing cytochrome b5 reductase 4 (CYB5R4) and enhanced green fluorescent protein (EGFP) using Lipofectamine 3000. Two STING inhibitors, MRT67307 and BX795, were added during transfection and overnight post-transfection. As a result, MRT67307 significantly enhanced CYB5R4 and EGFP expression, even 24 hours after its removal. In comparison, MRT67307 pretreatment did not affect transfection, suggesting the inhibitor's effect was readily reversible. The phosphorylation of endothelial nitric oxide synthase (eNOS) at Serine 1177 (S1177) by vascular endothelial growth factor is essential for endothelial proliferation, migration, and survival. Using the same protocol, we transfected wild-type and phosphorylation-incapable mutant (S1177A) eNOS in HAECs. Both forms of eNOS localized on the plasma membrane, but only the wild-type eNOS was phosphorylated by vascular endothelial growth factor treatment, indicating normal functionality of overexpressed proteins. MRT67307 and BX795 also improved plasmid expression in human and rat aortic SMCs. In conclusion, this study presents a modification enabling efficient plasmid transfection in primary vascular ECs and SMCs, offering a favorable approach to studying protein function(s) in these cell types, with potential implications for other primary cell types that are challenging to transfect.

Green synthesis of silver nanoparticles: understanding biological activities – anticancer, antimicrobial, and plasmid DNA cleavage properties through reactive oxygen species release

Study on green-synthesized nanoparticles: preparation, characterization, biological effects, and DNA degradation capabilities confirmed through UV-visible and FTIR analysis.

SNP genotyping and environmental niche modelling using herbarium specimens of the northern dragonhead, Dracocephalum ruyschiana (Lamiaceae)

Maintenance of genetic diversity is a central aim of species conservation, given its positive effect on species survival and adaptation in a changing environment. Data from different time points is key for understanding how populations behave under various conditions. In this regard, herbarium specimens are an invaluable source of information from the past. Still, utilizing archived biological material for studying trends of genetic diversity offers challenges such as DNA degradation and the lack of standardized, cost- and time efficient methods.

Herbarium DNA degradation – Falling to pieces non-randomly

Post-mortem damage in herbarium DNA, mostly from 18th and 19th century collections, and with specimens usually heat-treated for conservation, consists mainly of genome fragmentation (single- and double-stranded breaks) rather than miscoding lesions. With typical herbarium DNA fragment sizes encountered (20–200 6 https://www.jstor.org/stable/1220096 bp) this easily leads to insert sizes in library construction being smaller than Illumina read lengths applied (i.e. 100–250 bp). Using a previously-published series of 56 genome-skimmed herbarium DNA extracts representing 10 angiosperm families, overlapping read pairs were found to occur in roughly 80 % of all read pairs obtained. After merging such overlapping pairs, the resulting fragments and their length-distributions are considered to reflect actual DNA fragmentation. Similar to occurrence in ancient DNA, we found over-representation of purines at fragment-ends in herbarium material. Distributions of fragment lengths fit gamma rather than exponential distributions, without apparent correlation with specimen age. The observed gamma distributions would indicate higher-order degradation kinetics, implying multiple processes acting during degradation. Possibly, the genome skimming data used here, in which repetitive sequences or compartments are over-represented, has biased genomic fragment-length distributions and half-lives as compared to the non-repetitive fraction of plant genomes, but no data was available to test this hypothesis. Overall, our results imply that we cannot confirm whether a plant archival DNA half-live exists and what its rate would be.

Copper(II) Complexes with 1-(Isoquinolin-3-yl)heteroalkyl-2-ones: Synthesis, Structure and Evaluation of Anticancer, Antimicrobial and Antioxidant Potential.

Four copper(II) complexes, C1-4, derived from 1-(isoquinolin-3-yl)heteroalkyl-2-one ligands L1-4 were synthesized and characterized using an elemental analysis, IR spectroscopic data as well as single crystal X-ray diffraction data for complex C1. The stability of complexes C1-4 under conditions mimicking the physiological environment was estimated using UV-Vis spectrophotometry. The antiproliferative activity of both ligands L1-4 and copper(II) compounds C1-4 were evaluated using an MTT assay on four human cancer cell lines, A375 (melanoma), HepG2 (hepatoma), LS-180 (colon cancer) and T98G (glioblastoma), and a non-cancerous cell line, CCD-1059Sk (human normal skin fibroblasts). Complexes C1-4 showed greater potency against HepG2, LS180 and T98G cancer cell lines than etoposide (IC50 = 5.04-14.89 μg/mL vs. IC50 = 43.21->100 μg/mL), while free ligands L1-4 remained inactive in all cell lines. The prominent copper(II) compound C2 appeared to be more selective towards cancer cells compared with normal cells than compounds C1, C3 and C4. The treatment of HepG2 and T98G cells with complex C2 resulted in sub-G1 and G2/M cell cycle arrest, respectively, which was accompanied by DNA degradation. Moreover, the non-cytotoxic doses of C2 synergistically enhanced the cytotoxic effects of chemotherapeutic drugs, including etoposide, 5-fluorouracil and temozolomide, in HepG2 and T98G cells. The antimicrobial activities of ligands L2-4 and their copper(II) complexes C2-4 were evaluated using different types of Gram-positive bacteria, Gram-negative bacteria and yeast species. No correlation was found between the results of the antiproliferative and antimicrobial experiments. The antioxidant activities of all compounds were determined using the DPPH and ABTS radical scavenging methods. Antiradical tests revealed that among the investigated compounds, copper(II) complex C4 possessed the strongest antioxidant properties. Finally, the ADME technique was used to determine the physicochemical and drug-likeness properties of the obtained complexes.

Molecular mechanism of allosteric activation of the CRISPR ribonuclease Csm6 by cyclic tetra-adenylate

Type III CRISPR systems are innate immune systems found in bacteria and archaea, which produce cyclic oligoadenylate (cOA) second messengers in response to viral infections. In these systems, Csm6 proteins serve as ancillary nucleases that degrade single-stranded RNA (ssRNA) upon activation by cOA. In addition, Csm6 proteins also possess cOA-degrading activity as an intrinsic off-switch to avoid degradation of host RNA and DNA that would eventually lead to cell dormancy or cell death. Here, we present the crystal structures of Thermus thermophilus (Tt) Csm6 alone, and in complex with cyclic tetra-adenylate (cA4) in both pre- and post-cleavage states. These structures establish the molecular basis of the long-range allosteric activation of TtCsm6 ribonuclease by cA4. cA4 binding induces significant conformational changes, including closure of the CARF domain, dimerization of the HTH domain, and reorganization of the R-X4-6-H motif within the HEPN domain. The cleavage of cA4 by the CARF domain restores each domain to a conformation similar to its apo state. Furthermore, we have identified hyperactive TtCsm6 variants that exhibit sustained cA4-activated RNase activity, showing great promise for their applications in genome editing and diagnostics.

Simple and Practical DNA Quantification Method for DNA-Encoded Library Synthesis.

Over the past three decades, DNA-encoded library (DEL) technologies have become one of the most relevant strategies for hit-finding. Recent advances in synthetic methodologies for DNA-encoded libraries rendered the increased chemical space available, but it is unknown how every variety of chemistry affects DNA's integrity. Available assays to quantify DNA damage are restricted to electrophoresis, ligation efficiency, and mostly qPCR quantification and sequencing, which may contain predisposition and inconsistency. We developed an external standard method through LC-MS analysis to accurately quantify DNA damage throughout the chemical transformations. An assessment was conducted on on-DNA chemical reactions that are frequently employed in DEL synthesis, and these results were compared to traditional qPCR measurements. Our study provides a simple, practicable, and accurate measurement for DNA degradation during DEL synthesis. Our finding reveals substantial disagreement among the usual DNA-damaging assessment methods, which have been largely neglected so far.

Enhanced in-vitro anti-Candida efficacy of Euphorbia milii Des Moul mediated copper nanoparticles against clinically isolated Candida albicans.

Emergence of fungi as a pathogenic threat presents a significant challenge to public health, notably in intensive care units (ICUs) and among immunocompromised patients. Various factors, including sepsis-induced barrier disruptions, immune system dysfunction, and extremes of age, contribute to increased susceptibility to fungal infections. Hospital practices, such as prolonged surgeries, broad-spectrum antibiotic use, and invasive procedures, further exacerbate the risk. Fungal bloodstream infections, particularly those caused by Candida albicans, rank among the most common hospital-acquired infections, leading to substantial morbidity and mortality. The global rise in invasive candidiasis, particularly due to non-albicans Candida species, presents challenges in the diagnosis and treatment due to nonspecific symptoms and emerging antifungal resistance. Nanotechnology interventions particularly by utilizing green synthesized copper nanoparticles could possibly provide a novel solution to combat microbial colonization, biofilm formation, and drug resistance. This study aimed to assess the prevalence of candidemia, identify the distribution of causative Candida species, and understand their susceptibility patterns to commonly used antifungal agents for effective management in ICU settings. Additionally, the study sought to explore the in vitro anti-Candida activity of green copper nanoparticles synthesized using Euphorbia milii des moul extract. This study was conducted at Microbiology Laboratory of Maharishi Markandeshwar Institute of Medical Sciences and Research from January to December 2022, focused on ICU patients suspected of bloodstream infections. Blood samples were collected aseptically and processed using BD BACTECTM culture vials. Identification of organisms was performed via the Vitek-2 system by confirming candidemia with positivity in both blood samples. After that antifungal susceptibility testing was also performed against Clinical and Laboratory Standards Institute recommended antifungal drug using Vitek 2 system. G-CuNPs were synthesized using E. milii Des moul extract and possessed for physiochemical characterization. The anti-Candida activity of G-CuNPs was evaluated through the MTT assay and time kill assay. After that generation of intracellular reactive oxygen species and DNA degradation were evaluated to understand its mechanism. This study identified a candidemia rate of 7.3% (58/789). Age and gender analysis revealed higher Candida colonization rates in individuals above 60 years old and females. Antifungal sensitivity profiling indicated notable resistance to fluconazole (27.59%) and voriconazole (25.86%). Synthesizing G-CuNPs using E. milii des moul extract represents a novel approach exhibiting significant fungicidal potency against clinically isolated C. albicans, supporting potential therapeutic applications. the findings concluded that synthesized G-CuNPs have tremendous potential to battle against medical device-borne infections by surface coating.

Performance of environmental DNA metabarcoding to identify and quantify benthic organisms in river ecosystems

Our lack of fundamental knowledge about species occurrence, abundance, distribution, threats, and habitat requirements makes it challenging to conserve biodiversity in the face of rising human pressure. To obtain this information, practical and sensitive methods are required to identify and quantify the occurrence and diversity of rare, cryptic, and elusive species. Environmental DNA (eDNA) is a new technology that can help to discover and quantify biodiversity by addressing some of the limitations of conventional surveys. This study aimed to evaluate the biodiversity and composition of benthic species in the Lower Ciliwung River, Jakarta, Indonesia. The investigation was conducted from upstream to downstream of the river at the three sites during the dry season (July 2022). The DNA read sequences were used to examine the composition, relative abundance, and diversity of the samples. In this study, 1,492,975 initial reads were filtered down to 1,265,307 reads belonging to 27 species of benthic organisms. The taxa identified from the eDNA samples revealed that the most frequently detected species belonged to the family Siboglinidae, order Sabellida, and class Polychaeta. The composition of benthic species discovered in the eDNA water samples varied substantially (p ≤ 0.05). Osedax sp. (class Polychaeta) was the most prevalent species across all locations. The diversity index (H) ranged from low to moderate. eDNA offers potential advantages, but is hampered by DNA degradation, primer sensitivity, and the inability to distinguish between dead and living organisms.

Accurate Detection of Multiple Tumor Mutations in Formalin-Fixed Paraffin-Embedded Tissues by Coupling Sequence Artifacts Elimination and Mutation Enrichment With MeltArray

Formalin-fixed paraffin-embedded (FFPE) tissues are the primary source of DNA for companion diagnostics (CDx) of cancers. Degradation of FFPE tissue DNA and inherent tumor heterogeneity constitute serious challenges in current CDx assays. To address these limitations, we introduced sequence artifact elimination and mutation enrichment to MeltArray, a highly multiplexed PCR approach, to establish an integrated protocol that provides accuracy, ease of use, and rapidness. Using PIK3CA mutations as a model, we established a MeltArray protocol that could eliminate sequence artifacts completely and enrich mutations from 23.5- to 59.4-fold via a single-reaction pretreatment step comprising uracil-DNA-glycosylase excision and PCR clamping. The entire protocol could identify 13 PIK3CA hotspot mutations of 0.05% to 0.5% mutant allele fractions within 5 hours. Evaluation of 106 breast cancer and 40 matched normal FFPE tissue samples showed that all 47 PIK3CA mutant samples were from the cancer tissue, and no false-positive results were detected in the normal samples. Further evaluation of 105 colorectal and 40 matched normal FFPE tissue samples revealed that 11 PIK3CA mutants were solely from the cancer sample. The detection results of our protocol were consistent with those of the droplet digital PCR assays that underwent sequence artifact elimination. Of the 60 colorectal samples with next-generation sequencing results, the MeltArray protocol detected 2 additional mutant samples with low mutant allele fractions. We conclude that the new protocol provides an improved alternative to current CDx assays for detecting tumor mutations in FFPE tissue DNA.