Amount Of DNA Research Articles

Evaluation of DNA Extraction Methods for Microbial Community Profiling in Deadwood Decomposition.

As technologies advance alongside metabarcoding and metagenomic resources, particularly for larger fungal genomes, DNA extraction methods must be optimized to meet higher thresholds, especially from complex environmental substrates. This study focused on extracting fungal genomic compounds from woody substrates, a challenge due to the embedment of endophytic and saprotrophic fungi within wood cells, the physical recalcitrance of wood, the adsorption of nucleic acids to wood polymers, and the release of downstream inhibitors. Hypothesizing that cetyltrimethylammonium bromide would be the best option, we compared prominent methods by extracting and sequencing microbial DNA from sound and decayed birch (Betula papyrifera) and pine (Pinus resinosa). DNA quantities varied significantly depending on extraction methods and decay stage. The quality of DNA, in terms of purity and integrity, significantly impacted whether the samples could be amplified and sequenced. However, amplicon sequencing of bacterial and fungal communities revealed no significant extraction bias. This, along with the sequencing effectiveness and cost/time efficiency, indicates that Qiagen is the gold standard for woody substrates. This study increases confidence in published amplicon data sets regardless of the extraction methods, provides a cost-benefit table for making protocol decisions, and offers guidance on fungal DNA extractions from complex organic substrates (sound and decayed wood) that would best suit future metagenomic efforts.

Comprehensive Evaluation of Decellularized Dental Pulp as a New Biomaterial for Regeneration of the Pulp-Dentin Complex

Objective: To develop a detergent-enzymatic method and evaluate the quality of a decellularized pulp scaffold for regenerative endodontics.Materials and methods: Biomaterial and mesenchymal stem cells (MSCs) were derived from dental pulp that was obtained following third molar extraction indicated for orthodontic reasons in patients aged 14-18 years. The detergent-enzymatic method enabled to obtain a decellularized scaffold from pulp samples. The proliferative activity and viability of dental pulp-derived MSCs were assessed using trypan blue staining and XTT assay. To assess tissue response, Wistar rats underwent subcutaneous implantation of native and decellularized dental pulp. Explanted samples were stained with hematoxylin-eosin on days 7 and 14.Results: The detergent-enzymatic treatment of the dental pulp demonstrated the absence of nuclear material, whereas the histoarchitecture of the dental pulp was disturbed. The DNA content in the sample of the decellularized scaffold was 22.79 ± 2.1 ng/mg of tissue; the amount of DNA in the native sample was 78.5 ± 5.4 ng/mg of tissue. According to XTT assay results, no cytotoxicity of the decellularized scaffold against MSCs was found. Biopsy specimens of the rats with implanted decellularized dental pulp were characterized by no signs of inflammation.Conclusions: The study results will enable to create a biomaterial that can be the base of a tissue-engineered structure of the dental pulp and be used for the regeneration of the pulp-dentin complex.

Circulating levels of Low Density Granulocytes and cell-free DNA as predictors of cardiovascular disease and bone deterioration in SLE patients.

The present work evaluates the predictive value of low-density-granulocytes (LDG) for the development of cardiovascular disease (CVD) and/or bone deterioration (BD) in a six-year prospective study in Systemic Lupus Erythematosus (SLE). Considering the high SLE-LDG capacity to form Neutrophil Extracellular Traps (NETs), circulating levels of total cell-free DNA (cirDNA) and relative amounts of mitochondrial and nuclear DNA (mtDNA and nDNA, respectively) were tested as LDG-associated biomarkers to identify SLE patients at risk of CVD and BD. To this end, the frequency of total blood LDGs, as well as the CD16negCD14neg (nLDG) and CD16posCD14low (pLDG) subsets, was quantified by flow cytometry in 33 controls and 144 SLE patients. Total cirDNA and relative amounts of mitochondrial (mtDNA) and nuclear (nDNA) cell-free DNA were measured by fluorometry or qPCR in plasma from a subgroup of 117 patients and 23 controls at enrolment. Our findings showed increased blood levels of SLE-nLDGs at enrolment associated with prospective CVD development (pCVD) and the presence of BD, thus revealing LDG expansion as a predictor of both comorbidities in SLE progression. The amounts of the different types of circulating DNA analysed were increased in patients, especially those presenting traditional CV-risk factors or subclinical atheromatosis. Similar to nLDGs, the nDNA concentration could predict the development of pCVD in SLE, supporting the quantification of cirDNA levels as a surrogate marker of LDGs in clinical practice.

Comparative evaluation of commercial DNA isolation approaches for nanopore-only bacterial genome assembly and plasmid recovery.

The advent of Oxford Nanopore Technologies has undergone significant improvements in terms of sequencing costs, accuracy, and sequencing read lengths, making it a cost-effective, and readily accessible approach for analyzing microbial genomes. A major challenge for bacterial whole genome sequencing by Nanopore technology is the requirement for a higher quality and quantity of high molecular weight DNA compared to short-read sequencing platforms. In this study, using eight pathogenic bacteria, we evaluated the quality, quantity, and fragmented size distribution of extracted DNA obtained from three different commercial DNA extraction kits, and one automated robotic platform. Our results demonstrated significant variation in DNA yield and purity among the extraction kits. The ZymoBIOMICS DNA Miniprep Kit (ZM) provided a higher purity of DNA compared to other kit-based extractions. All kit-based DNA extractions were successfully performed on all twenty-four samples using a single MinION flow cell, with the Nanobind CBB Big DNA kit (NB) yielding the longest raw reads. The Fire Monkey HMW-DNA Extraction Kit (FM) and the automated Roche MagNaPure 96 platform (RO) outperformed in genome assembly, particularly in gram-negative bacteria. Based on our finding, we recommend a minimum read coverage and raw read N50, obtained from the appropriate DNA extraction kit for each bacterial species, to optimize genome assembly and plasmid recovery. This approach will assist end-users in selecting the most effective kit-based extraction method for bacterial whole-genome assembly using only long-read nanopore sequences.

Abstract 4132149: Elevated Levels of Plasma Mitochondrial DNA Damage-Associated Molecular Patterns (DAMPS) in Humans and Dogs with Heart Failure and Reduced Ejection Fraction

Background: Heart failure (HF) with reduced ejection fraction (HFrEF) is associated with increased inflammatory response that contributes to progressive worsening of the HF state. Potential endogenous triggers of this process include damage-associated molecular patterns (DAMPs). DAMPs originate from within cells following tissue stress or injury and activate pattern recognition receptors of the immune system, triggering an inflammatory response. Mitochondria are the major cell organelles that release DAMPs into the circulation. Chronic HF is associated with multi-organ mitochondrial dysfunction and cell injury and death. Purpose: The present study sought to quantify differences in mitochondrial DNA (mtDNA) DAMPs in the setting of HFrEF in humans and dogs compared to normal humans and dogs. Methods: Plasma samples were obtained from 9 HFrEF patients with left ventricular ejection fraction (LVEF) ≤35% and 9 age-matched healthy subjects, and from 6 dogs with coronary microembolization-induced HF (LVEF ≤35%) and 6 normal dogs. DNA fragments were isolated from 1 ml of plasma using a commercially available kit. Quantitative PCR and specifically designed primers were used to measure the amount of DNA in plasma from COX1 (cytochrome c oxidase subunit 1), ND1 (NADH dehydrogenase subunit 1) and ND6 (NADH dehydrogenase subunit 6) using an Applied Biosystems 7500 Fast-PCR unit. The relative abundances of plasma mtDNA DAMPs were expressed as threshold cycles (C T ). Results: Compared to normal human subjects, mtDNA DAMPs levels of COX1 , ND1 and ND6 in plasma of HFrEF patients was 4-fold, 9-fold and 8-fold greater, respectively. Similarly, compared to normal dogs, mtDNA DAMPs levels of COX1 , ND1 and ND6 in plasma of HFrEF dogs was increased 48-fold, 12-fold and 4-fold, respectively. Conclusions: These findings indicate a marked increase of mtDNA DAMPs in plasma of humans and dogs with HFrEF. The increase of mtDNA DAMPs is consistent with the elevated systemic inflammatory state of HFrEF. The results underscore mitochondrial abnormalities as integral players in the progressive worsening of HFrEF.

Rapid diagnosis of Mycoplasma pneumoniae and prediction of antibiotic resistance by nanopore adaptive sampling

AbstractBackgroundMicrobial infections, particularly in children, require rapid and accurate diagnostics. It is difficult to differentiate pathogens from commensal organisms, and it is impossible to identify antibiotic resistance genes that belong to pathogens with current methods. Third‐generation sequencing provides rapid library preparation and real‐time data acquisition. Nanopore normal sampling (NNS) enables unbiased sequencing of clinical samples without amplification, aiding pathogen identification and antimicrobial resistance gene prediction. However, clinical samples often contain a considerable amount of human DNA, potentially masking pathogen data. Nanopore adaptive sampling (NAS) aims to selectively enrich pathogens, promising improved diagnostics for acute infections and better treatment decisions in clinical practice. This study aimed to determine the utility of NAS in enhancing the real‐time detection of pathogens and predicting AMR in infectious disease outbreaks.MethodsThis study used NAS technology to rapidly and directly detect Mycoplasma pneumoniae infection in bronchoalveolar lavage fluid samples from 28 pediatric patients at Shenzhen Children's Hospital. We assessed the efficacy of NAS compared with that of NNS by evaluating the number of microbial reads and the amount of microbial DNA data. We then compared the accuracy of detecting pathogens between NNS and NAS and between NAS and real‐time polymerase chain reaction assays. Furthermore, we predicted antimicrobial resistance (AMR) and examined AMR genes associated with pathogens.ResultsNAS showed up to a 14.67‐fold increase in the amount of microbial DNA data from patients' samples compared with NNS within the initial 2.5 h of sequencing. Additionally, NAS reduced the amount of host DNA data by up to 6.67‐fold compared with NNS. Unlike TaqMan real‐time polymerase chain reaction assays, NAS technology identified dominant pathogens and provided detailed insight into the abundance of the microbial community. Furthermore, NAS was able to predict AMR profiles of microbial communities and attribute specific AMR traits to individual microbes within the samples.ConclusionThis study shows that NAS advances the clinical diagnosis because it can rapidly detect pathogens directly from patients' samples and provides antimicrobial resistance information for clinical guidance. These abilities further facilitate the application of NAS in personalized treatment, reduce the misuse of broad‐spectrum antibiotics, and promote patients' recovery.

QLTI-09. CLINICAL TRIALS IN NEURO-ONCOLOGY: DESIGN, LOGISTICS, CHALLENGES, AND PRACTICAL CONSIDERATIONS

Abstract Timely, secure and reliable transportation, precise handling and storage of biological specimens are critical for the success of clinical research. Research in neuro-oncology often involves complex assays such as next-generation sequencing where the integrityof brain tumor specimens is paramount. These challenges are pronounced in most Lower Middle-Income Countries (LMIC), due to climatic factors and infrastructure limitations, necessitating stringent measures to preserve sample quality and their genetic data integrity for downstream analysis. In this study, we conducted extensive cohort analysis on 80 tissues randomly picked samples of the 3600 samples collected across India to assess the impact of collection protocols, transportation logistics, and long-term storage on the genetic material of tissues. The quality and quantity of the isolated nucleic acids was determined by BioAnalyzer and Qubit parameters. Tissue samples were collected and stored at -80°C in RNAstable® within 4-12 hrs of collection. The DNA and RNA obtained from tissue samples stored for over one year was found to be of good quality with corresponding DNA Integrity Number (DIN) and RNA Integrity Number (RIN)between 7-9. Also, optimum quantity of DNA and RNA was obtained for whole exome and transcriptome sequencing. Upon sequencing, these samples showed good read coverage of 100X or more. These findings provide critical insights into the measures necessary for optimizing sample preservation and transportation to ensure accurate research outcomes. The results indicate that appropriate sample collection, transportation, and long-term storage of bio-specimens can be used effectively for future research. Additionally, the outcome underscores the potential for long-term studies in neuro-oncology using archived biological samples without compromising data integrity.

Air DNA forensics: Novel air collection method investigations for human DNA identification.

Modern techniques can generate highly discriminatory DNA profiles from minuscule biological samples, providing valuable information in criminal investigations and court proceedings. However, trace and touch DNA samples, due to their nature, often have lower success rates than other biological materials, such as blood. Further, forensically aware criminals can utilize gloves and meticulously clean the crime scene to remove DNA traces of themselves from contacted surfaces. Air sampling offers a novel approach to the collection of human DNA that has the potential to bypass some of these issues. This study reports on the results of research into the prevalence and persistence of human DNA in the air. The ability to collect human DNA from the air was investigated with the use of an AirPrep Cub Sampler ACD220 in different spaces, with and without the presence of individuals for various durations of sample collection. Results of this study demonstrate that level of occupation and sampling duration each have an influence on quantity and quality of DNA recovered from the air whereas the effects of orientation and distance of participants from the collection device as well as sequence of occupation remain unclear and require further investigation.

Patellae as a source of DNA in forensic and archaeological analysis.

Analysing genetic material from skeletonised human remains has become valuable in forensic and archaeological contexts. While the petrous bone is often preferred for DNA extraction, its availability is not guaranteed, and because of destructive sampling, it is not frequently used in forensic cases. This study explores the potential of patellae as an alternative source of bone material for genetic investigations. Forty-five patellae were sampled from a post-World War II mass grave and an archaeological Christian cemetery dated from the 13th to 19th centuries. A full demineralisation extraction method was used to obtain the DNA, and real-time PCR quantification was used to determine the quantity and quality of DNA. To evaluate the suitability of patellae for forensic and archaeological analyses, short tandem repeat (STR) typing was performed using the ESI17 Fast PCR amplification kit (Promega). To explore the difference in DNA yield, DNA degradation and STR typing success between the post-World War II and archaeological patellae, statistical analysis was performed. The results revealed significantly higher DNA yield and STR typing success in WWII patellae and higher degradation of DNA in archaeological patellae, highlighting the impact of environmental exposure time on genetic material preservation. Almost all WWII patellae achieved a high success rate in STR typing with full profiles generated. More than half of the archaeological patellae showed high STR typing performance and highly informative partial profiles were obtained, indicating the suitability of patellae not only for forensic purposes but also for archaeological genetic analyses.

How to best assess shedder status: a comparison of popular shedder tests.

"Shedder status" describes the inherent variation between individuals to leave touch DNA on a surface through direct contact. Depending on the amount and quality of DNA or cellular deposition, individuals are typically deemed high, intermediate, or low shedders. Although many shedder tests have been described, variability in study design and categorisation criteria has limited the ability of researchers to accurately compare results, as well as accrue the necessary population data. As activity level reporting becomes more common, the need for reliable and standardised testing increases. To assess reproducibility, this study compared shedder status data generated by six participants using three different shedder tests, as modified from the literature. This involved DNA quantification and profiling of a handprint made on a glass plate, DNA quantification and profiling of a grip mark made on a plastic conical tube, and cell scoring of a Diamond™ Dye-stained fingermark. All participants washed and dried their hands fifteen minutes before each deposit. To assess the impact of behaviour on shedder designation, participants either refrained from activity or went about their daily tasks during this wait. The shedder status of participants changed between tests, as DNA-based testing often generated lower shedder statuses than cell scores. Further, when different categorisation methods were applied to a single test, intra-person variability increased as the number of shedder designations increased from two (low/high) to five (low/low-intermediate/intermediate/intermediate-high/high). Moving forward, the utilisation of a single shedder test and standardised categorisation criteria is needed to employ shedder testing in forensic casework.

A Comparative Study of Methyl-BEAMing and Droplet Digital PCR for MGMT Gene Promoter Hypermethylation Detection

Background: O-6-methylguanine-DNA methyltransferase is responsible for the direct repair of O6-methylguanine lesions induced by alkylating agents, including temozolomide. O-6-methylguanine-DNA methyltransferase promoter hypermethylation is a well-established biomarker for temozolomide response in glioblastoma patients, also correlated with therapeutic response in colorectal cancer. Objectives: The ARETHUSA clinical trial aims to stratify colorectal cancer patients based on their mismatch repair status. Mismatch repair-deficient patients are eligible for treatment with immune checkpoint inhibitors (anti-PDL-1), whereas mismatch repair-proficient samples are screened for O-6-methylguanine-DNA methyltransferase promoter methylation to identify those suitable for temozolomide treatment. Methods: In this context, a subset of ARETHUSA metastatic colorectal cancer samples was used to compare two different techniques for assessing O-6-methylguanine-DNA methyltransferase hypermethylation: Methyl-BEAMing, a highly sensitive digital PCR approach that combines emulsion PCR and flow cytometry, and droplet digital PCR, a more automated procedure that enables the rapid, operator-independent analysis of a large number of samples. Results: Our study clearly demonstrates that the results obtained using Methyl-BEAMing and droplet digital PCR are comparable, with both techniques showing similar accuracy, sensitivity, and reproducibility. Conclusions: Digital droplet PCR proved to be an efficient method for detecting gene promoter methylation. However, the Methyl-BEAMing method has proved more sensitive for detecting low quantities of DNA.

Emerging threat of artemisinin partial resistance markers (pfk13 mutations) in Plasmodium falciparum parasite populations in multiple geographical locations in high transmission regions of Uganda

BackgroundArtemisinin-based combination therapy (ACT) is currently recommended for treatment of uncomplicated malaria. However, the emergence and spread of partial artemisinin resistance threatens their effectiveness for malaria treatment in sub-Saharan Africa where the burden of malaria is highest. Early detection and reporting of validated molecular markers (pfk13 mutations) in Plasmodium falciparum is useful for tracking the emergence and spread of partial artemisinin resistance to inform containment efforts.MethodsGenomic surveillance was conducted at 50 surveillance sites across four regions of Uganda in Karamoja, Lango, Acholi and West Nile from June 2021 to August 2023. Symptomatic malaria suspected patients were recruited and screened for presence of parasites. In addition, dried blood spots (DBS) were collected for parasite genomic analysis with PCR and sequencing. Out of 563 available dried blood spots (DBS), a random subset of 240 P. falciparum mono-infections, confirmed by a multiplex PCR were selected and used for detecting the pfk13 mutations by Sanger sequencing using Big Dye Terminator method. Regional variations in the proportions of pfk13 mutations were assessed using the chi square or Fisher’s exact tests while Kruskal–Wallis test was used to compare absolute parasite DNA levels between wild type and mutant parasites.ResultsOverall, 238/240 samples (99.2%) contained sufficient DNA and were successfully sequenced. Three mutations were identified within the sequenced samples; pfk13 C469Y in 32/238 (13.5%) samples, pfk13 A675V in 14/238 (5.9%) and pfk13 S522C in (1/238 (0.42%) samples across the four surveyed regions. The prevalence of pfk13 C469Y mutation was significantly higher in Karamoja region (23.3%) compared to other regions, P = 0.007. The majority of parasite isolates circulating in West Nile are of wild type (98.3), P = 0.002. Relative parasite DNA quantity did not differ in samples carrying the wild type, C469Y and A675V alleles (Kruskal–Wallis test, P = 0.6373).ConclusionDetection of validated molecular markers of artemisinin partial resistance in multiple geographical locations in this setting provides additional evidence of emerging threat of artemisinin partial resistance in Uganda. In view of these findings, periodic genomic surveillance is recommended to detect and monitor levels of pfk13 mutations in other regions in parallel with TES to assess potential implication on delayed parasite clearance and associated treatment failure in this setting. Future studies should consider identification of potential drivers of artemisinin partial resistance in the different malaria transmission settings in Uganda.

Comparison of Four Different DNA Isolation Methods from MGIT Culture for Long-Read Whole Genome Sequencing of Mycobacterium tuberculosis

Background: Tuberculosis (TB) remains a global health challenge, particularly due to drug resistance and limitations in rapid diagnosis. Next-generation sequencing (NGS), especially long-read whole genome sequencing (WGS), shows promise for rapidly detecting TB and drug resistance, but it requires high-quality DNA, which is difficult to extract from Mycobacterium tuberculosis due to its complex cell wall. Objectives: This study evaluated four DNA isolation methods for extracting pure DNA from M. tuberculosis, aiming to standardize protocols for long-read WGS. Methods: Mycobacterium tuberculosis H37RV colonies were grown in BACTEC MGIT liquid medium. Two pellets were prepared as the initial material for the DNA extraction protocol: Pellets from 1 mL McFarland 2 suspensions and all growing colonies from two MGIT liquid cultures. Four DNA extraction methods were used: The cetyltrimethylammonium bromide (CTAB) method, GeneJET Genomic DNA Purification Kit, Quick-DNA Fecal/Soil Microbe Kit, and Genematrix Tissue/Bacterial DNA Purification Kit, with some modifications. DNA quality was assessed based on concentration, purity, and integrity. Results: Among the tested methods, the Quick-DNA Fecal/Soil Kit yielded approximately 85 ng/mL of DNA and a purity of 1.9 at 260/280 nm from the colonial pellet of two MGIT tubes. However, lower intact DNA [DNA integrity number (DIN) ~ 6.8] was obtained with this kit. The CTAB method provided the highest intact DNA (DIN ~ 9.5), although the purity of the DNA was not sufficient. Conclusions: Based on three repetitions of McF-2 and colonial pellet extractions, the Quick-DNA Fecal/Soil Kit yielded the highest DNA quantity and purity but showed lower integrity compared to other methods, indicating the need for adjustments. A pellet from two MGIT cultures (~ 100 µL) is suitable for long-read WGS with this kit. However, a larger sample size is required to generalize these findings. For effective long-read sequencing of M. tuberculosis, DNA extraction protocols must be optimized to balance yield, fragment size, and purity for accurate sequencing and drug resistance analysis.

Development of a qPCR assay for the quantification of canine autosomal DNA recovered from livestock attacks

The absence of a standardised method to quantify canine DNA recovered from livestock attacks leaves forensic providers without an important quality control step to help support their decision making. Typically used to normalise the amount of DNA for STR amplification, modern forensic DNA quantification approaches use qPCR of target genes and can also include an Internal Positive Controls (IPC) to determine the presence of PCR inhibitors. The co-amplification of livestock DNA alongside canine DNA has meant that previously developed qPCR methods are not suitable for use so a standardised approach is needed. This research describes the development of a Taq-man multiplex qPCR assay that simultaneously quantifies the autosomal MC1R and Y-specific SRY gene to determine the concentration of canine DNA recovered from attacked livestock. Data suggests that the method is robust and reproducible with no significant difference in the standard curves produced from multiple runs or from different DNA standards derived from different canines. Assay sensitivity of between 15 and 31 pg is consistent with other forensic quantification assays and also in line with the sensitivity of the two tested canine STR kits, Canine Genotype 2.1 Kit and CaDNAP Panels 1 and 2. The assay is highly specific to canines when tested against 163 different dogs representing 33 different breeds and no cross-amplification of non-target species’ DNA was observed even from livestock DNA tested at 31.25ng/µl. This strongly suggests that any DNA detected on evidence collected from attacked livestock is canine. The assay also shows robust tolerance to common livestock inhibitors continuing to amplify when inhibitor-spiked DNA samples were tested. Both mixed and inhibited DNA samples underwent STR typing using two canine forensic STR kits with data showing the Canine Genotype 2.1 Kit displaying pronounced cross-amplification of livestock DNA and and/or extensive PCR inhibition leading to the complete loss of amplification when using this kit. Conversely the CaDNAP Panels 1 and 2 showed little cross-amplification of livestock DNA and improved inhibitor tolerance suggesting that this approach was better suited for the analysis of livestock attack samples. Findings are discussed and the impact of the observations on future work in this area are explored.

Surveillance of Disease Progression in Metastatic Breast Cancer by Molecular Counting of Circulating Tumor DNA Using Plasma-SeqSensei Breast Cancer in Vitro Diagnostics Assay

Circulating tumor DNA (ctDNA) quantification surpasses cancer antigen 15 to 3 for metastatic breast cancer surveillance. Clinical translation, however, is limited because of uncertainties about the optimal method and clinically valid ctDNA decision thresholds. Plasma-SeqSensei Breast Cancer IVD kit (PSS) is a novel assay for ctDNA molecular counting, detecting ≥0.06% variant allele fractions in AKT1, ERBB2, ESR1, KRAS, PIK3CA, and TP53. PSS was validated against droplet digital PCR (ddPCR) in 201 samples from 16 subjects with PIK3CA/TP53-mutated cancers, longitudinally sampled for a median of 93 (range, 18 to 113) weeks, three to five weekly. PSS and ddPCR ctDNA levels correlate significantly (Spearman ρ, 0.923; 95% CI, 0.898-0.941) across 0% to 43% variant allele frequency (VAF) range. PSS predicts 12-week progression with high clinical accuracy (area under the curve, 0.848; 95% CI, 0.790-0.894). PSS validates a previously developed ddPCR classifier: <10 copies/mL (0.25% VAF); excludes >100 copies/mL (2.5% VAF); and confirms progression, with negative predictive value (95% CI) of 83% (76%-88%) and positive predictive value (95% CI) of 91% (81%-96%) (weighted κ, 0.856; 95% CI, 0.797-0.915). PSS thus confirms robust clinical thresholds (10 to 100 copies/mL, 0.25% to 2.5% VAF) for metastatic breast cancer surveillance, using absolute molecular counting.

Characterization of Extracellular Matrix Derived From Porcine Organs Decellularized Using Different Methods.

Regenerative medicine has extended the capacity of medicine to a point where tissues and organs could potentially be manufactured. This could resolve issues associated with organ transplantation. The extracellular matrix (ECM) provides a supportive scaffold and biochemical cues allowing cells to attach, proliferate, and differentiate. The ECM is composed of different fibrous proteins and proteoglycans. The extensive value of ECM lies in its dynamic microenvironment that aids in cell proliferation, differentiation, and regulation of intercellular communication. In this study, four methods were applied to decellularize porcine organs. The ECMs were characterized by histological methods illustrating the absence of nuclei and the presence of glycosaminoglycans (GAGs) and collagen. Hematoxylin and eosin analysis of native pancreas revealed necrosis by auto-digestion, also supported by a reduced dsDNA content, and could have led to the destruction of Type IV collagen, laminins, and other proteins in the resulting ECMs, as confirmed by mass spectrometry. DNA quantification of ECM revealed residual dsDNA contents lower than those of the native organs. Bicinchoninic acid (BCA) assay showed a difference in protein content between organs. Mass spectrometry coupled with proteomic analysis highlighted a significant difference in the protein composition. The number of different proteins, in some cases with more than 2700, in the produced ECM depended on the applied decellularization technique. Polarization microscopy indicated differences in the orientation of collagen fibers. This study provides a multimodal approach to characterize ECMs produced using different decellularization techniques, aiding in finding a balance between maintaining the ultrastructure and composition of ECM, while removing cellular components.

Impact of sample processing delays on plasma markers of inflammation, chemotaxis, cell death, and blood coagulation.

Biosampling studies in critically ill patients traditionally involve bedside collection of samples followed by local processing (ie. centrifugation, aliquotting, and freezing) and storage. However, community hospitals, which care for the majority of Canadian patients, often lack the infrastructure for local processing and storage of specimens. A potential solution is a "simplified" biosampling protocol whereby blood samples are collected at the bedside and then shipped to a central site for processing and storage. One potential limitation of this approach is that delayed processing may alter sample characteristics. To determine whether delays in blood sample processing affect the stability of cytokines (IL-6, TNF, IL-10, IFN-γ), chemokines (IL-8, IP-10, MCP-1, MCP-4, MIP-1α, MIP-1β), cell-free DNA (cfDNA) (released by dying cells), and blood clotting potential in human blood samples. Venous blood was collected into EDTA and citrate sample tubes and stored at room temperature (RT) or 4°C for progressive intervals up to 72 hours, prior to processing. Plasma cytokines and chemokines were quantified using single or multiplex immunoassays. cfDNA was measured using Picogreen DNA Quantification. Blood clotting potential was measured using a thrombin generation assay. Blood samples were collected from 9 intensive care unit (ICU) patients and 7 healthy volunteers. Admission diagnoses for the ICU patients included sepsis, trauma, ruptured abdominal aortic aneurysm, intracranial hemorrhage, gastrointestinal bleed, and hyperkalemia. After pre-processing delays of up to 72 hours at RT or 4°C, no significant changes were observed in plasma cytokines, chemokines, cfDNA, or thrombin formation. Delayed sample processing for up to 72 hours at either RT or 4°C did not significantly affect cytokines, chemokines, cfDNA, or blood clotting potential in plasma samples from healthy volunteers and ICU patients. A "simplified" biosampling protocol is a feasible solution for conducting biosampling research at hospitals without local processing capacity.

Beyond the mix: Advancements in simultaneous detection and quantification of human, dog, and cat DNA.

Animal-related crimes have increased with an increase in the number of pets worldwide, underscoring the importance of animal-related biological evidence at crime scenes. Evidence obtained in cases involving dogs and cats often includes a mixture of human and animal DNA. In this study, we developed a method using droplet digital polymerase chain reaction (ddPCR) to simultaneously identify and quantitatively detect human, dog, and cat DNA in mixed samples. HLA-DRA was chosen as a human-specific marker, OR6D7 as a dog-specific marker, and FLAI-K as a cat-specific marker. The species specificity of each target was confirmed using 14 control DNA samples from 11 mammals and 3 birds. Sensitivity tests determined the limit of detection to be 0.0008 ng/μL for human DNA and 0.00061 ng/μL for dog and cat DNA. In the mixture test, each DNA sample was independently and accurately detected in samples containing trace amounts of all three types of DNA. This study demonstrated the effectiveness of applying ddPCR to forensic case samples from dog- and cat-related incidents. We have presented a reliable method for the accurate identification and quantification of human, dog, and cat DNA simultaneously, offering possibilities for advancements in forensic DNA analysis and related fields.

DNA extraction protocol from Myrciariacaerulescens J.M.A. Braga, M.T.C. Lacerda &amp; Stadnik (MYRTACEAE)

Myrciaria caerulescens, a fruit-bearing species endemic to Bahia, Brazil, and recently described, has ecological and economic potential. The analysis of genetic diversity and the use of molecular techniques are essential for its conservation and genetic improvement of M. caerulescens.The objective of this work was to standardize the protocol for extracting total DNA from Myrciaria caerulescens in the municipalities of Caetité and Lagoa Real in Bahia. Young leaves were collected using three collection methods and added to aluminum foil stored on ice, aluminum foil with silica and silica in a 50 mL test tube, where they received alphanumeric identification.The material was taken to the Genetic Improvement and Biotechnology Center Laboratory at UFRB, the silica was removed from the samples and stored in an ultra freezer at -80°C. For genomic DNA extraction, the three collection and extraction methods of the 40 samples, three protocols were tested for each method. The protocols of Lodhi (1994), modified by Pereira (2005), Doyle and Doyle (1987) and Murray and Tompsom (1980).Liquid nitrogen was used for crushing, helping to break down the M.caerulescens material. The isolated DNA influenced the quality and quantity of the methods and protocols tested. Silica gel method in the test tube had a greater amount of DNA compared to the others.The best result in DNA bands was described in Lodhi (1994), modified by Pereira (2005), better quality and higher concentration of extracted DNA, the test tube with silica, greater presence of bands, following the aluminum foil with silica for both locations. CTAB protocols were efficient in extraction. The increase in the amount of NaCl (0.5 to 5M) made it possible to quantify DNA bands in the agarose gel with positive ions neutralizing the negative charge of the DNA phosphate group.

Multiplexed Methylated DNA Immunoprecipitation Sequencing (Mx-MeDIP-Seq) to Study DNA Methylation Using Low Amounts of DNA

Background/Objectives: DNA methylation is a key epigenetic mark involved in regulating gene expression. Aberrant DNA methylation contributes to various human diseases, including cancer, autoimmune disorders, atherosclerosis, and cardiovascular diseases. While whole-genome bisulfite sequencing and methylated DNA immunoprecipitation (MeDIP) are standard techniques for studying DNA methylation, they are typically limited to a few samples per run, making them expensive and low-throughput. Therefore, an automation-friendly method is needed to increase throughput and reduce costs without compromising data quality. Methods and Results: We developed a novel method called Multiplexed Methylated DNA Immunoprecipitation Sequencing (Mx-MeDIP-Seq), which can be used to analyze many DNA samples in parallel, requiring only small amounts of input DNA. In this method, 10 different DNA samples were fragmented, purified, barcoded, and pooled prior to immunoprecipitation. In a head-to-head comparison, we observed a 99% correlation between MeDIP-Seq performed individually or combined as Mx-MeDIP-Seq. Moreover, multiplexed MeDIP led to more than 95% normalized percent recovery and a 25-fold enrichment ratio by qPCR, like the enrichment of the conventional method. This technique was successfully performed with as little as 25 ng of DNA, equivalent to 3400 to 6200 cells. Up to 10 different samples were processed simultaneously in a single run. Overall, the Mx-MeDIP-Seq method is cost-effective with faster processing to analyze DNA methylome, making this technique more suitable for high-throughput DNA methylome analysis. Conclusions: Mx-MeDIP-Seq is a cost-effective and efficient method for high-throughput DNA methylation analysis, offering faster processing and reduced sample requirements. This technique makes DNA methylome analysis more accessible for large-scale studies.