DNA Specimens Research Articles

Paraoxonase-1 as a Cardiovascular Biomarker in Caribbean Hispanic Patients Treated with Clopidogrel: Abundance and Functionality.

Clopidogrel, a prescription drug to reduce ischemic events in cardiovascular patients, has been extensively studied in mostly European individuals but not among Caribbean Hispanics. This study evaluated the low abundance and reduced activity of paraoxonase-1 (PON1) in clopidogrel-resistant patients as a predictive risk biomarker of poor responders and disease severity in this population. Thirty-six patients on clopidogrel (cases divided into poor and normal responders) were enrolled, along with 11 cardiovascular patients with no clopidogrel indications (positive control) and 13 healthy volunteers (negative control). Residual on-treatment platelet reactivity unit (PRU), PON1 abundance by Western blotting, and PON1 activity by enzymatic assays were measured. PON1 genotyping and computational haplotype phasing were performed on 512 DNA specimens for two genetic loci (rs662 and rs854560). No statistical differences in mean relative PON1 abundance were found among the groups (p > 0.05). However, a significantly lower enzymatic activity was found in poor responders (10.57 ± 6.79 µU/mL) when compared to controls (22.66 ± 8.30 µU/mL and 22.21 ± 9.66 µU/mL; p = 0.004). PON1 activity among carriers of the most prevalent PON1 haplotype (AA|AA) was significantly lower than in wild types (7.90 µU/mL vs. 22.03 µU/mL; p = 0.005). Our findings suggested that PON1 is a potential biomarker of cardiovascular disease severity in Caribbean Hispanics.

Repeated rebiopsy for detection of EGFR T790M mutation in patients with advanced-stage lung adenocarcinoma: Associated factors and treatment outcomes of Osimertinib.

This study was performed to investigate the detection rate of EGFR T790M mutation by repeated rebiopsy, to identify the clinical factors related to repeated rebiopsy, and to assess survival outcomes according to the methods and numbers of repeated rebiopsies in patients with lung adenocarcinoma who received sequential osimertinib after failure of previous 1st or 2nd generation EGFR-tyrosine kinase inhibitors. This retrospective study included patients with advanced-stage lung adenocarcinoma who were confirmed to have EGFR T790M mutation and to have received osimertinib from January 2020 to February 2021 at Samsung Medical Center. The presence of T790M mutation was assessed based on either plasma circulating tumor DNA (ctDNA) or tissue specimens. Results A total of 443 patients underwent rebiopsy, with 186 (42.0%) testing positive for the T790M mutation by the sixth rebiopsy. The final analysis included 143 eligible patients. Progression-free survival was not significantly different in terms of the methods (tissue: 13.3 months, 95% confidence interval [CI]: [9.4, 23.5] vs plasma: 11.1 months, 95% CI: [8.1, 19.4], p = 0.33) and numbers (one: 13.4 months, 95% CI: [9.4, 23.5] vs two or more: 11.0 months, 95% CI: [8.1, 14.8], p = 0.51) of repeated rebiopsies. Longer overall survival (OS) was found in patients in whom T790M was detected by tissue specimens rather than by plasma ctDNA (2-year OS rate: 81.7% for tissue vs 63.9% for plasma, p = 0.0038). Factors related to the lower numbers of rebiopsies included age and bone metastasis. Factor associated with T790M detection in tissue rather than in plasma was pleural metastasis, while advanced tumor stage was related to T790M confirmation in plasma rather than in tissue. Repeated rebiopsy for T790M detection in patients with NSCLC can increase the detection rate of the mutation. Detection of T790M by plasma ctDNA might be related to poor survival outcomes.

A PCR Test Using the Mini-PCR Platform and Simplified Product Detection Methods Is Highly Sensitive and Specific to Detect Fasciola hepatica DNA Mixed in Human Stool, Snail Tissue, and Water DNA Specimens.

Fasciola hepatica has a complex lifecycle with multiple intermediate and definitive hosts and influenced by environmental factors. The disease causes significant morbidity in children and its prevalent worldwide. There is lack of data about distribution and burden of the disease in endemic regions, owing to poor efficacy of the different diagnostic methods used. A novel PCR-based test was developed by using a portable mini-PCR® platform to detect Fasciola sp. DNA and interpret the results via a fluorescence viewer and smartphone image analyzer application. Human stool, snail tissue, and water samples were used to extract DNA. Primers targeting the ITS-1 of the 18S rDNA gene of Fasciola sp. were used. The limit of detection of the mini-PCR test was 1 fg/μL for DNA samples diluted in water, 10 fg/μL for Fasciola/snail DNA scramble, and 100 fg/μL for Fasciola/stool DNA scramble. The product detection by agarose gel, direct visualization, and image analyses showed the same sensitivity. The Fh mini-PCR had a sensitivity and specificity equivalent to real-time PCR using the same specimens. Testing was also done on infected human stool and snail tissue successfully. These experiments demonstrated that Fh mini-PCR is as sensitive and specific as real time PCR but without the use of expensive equipment and laboratory facilities. Further testing of multiple specimens with natural infection will provide evidence for feasibility of deployment to resource constrained laboratories.

Identification of Salmonella Enterica Serovar Typhi DNA by loop-mediated isothermal amplification with fluorescent detection

Introduction. Real-time PCR may be used along with loop isothermal amplification method (LAMP) allowing to conduct the study in 30–40 minutes to diagnose typhoid fever. Several LAMP assay variations for detecting Salmonella enterica serovar Typhi have been described. The studies report that relevant primers were tested on strains specific for Malaysia and China. We attempted to evaluate the LAMP primers described above for identifying S. Typhi strains specific for the Russian Federation and compare their sensitivity and specificity with each other. Materials and methods. A comparative in silico analysis of target sequences was carried out both in the open NCBI database and among the genetic sequences of collection strains at the St. Petersburg Pasteur Institute. Several sets of primers for LAMP amplification of various S. Typhi genome regions were tested. The tested primers amplify the following fragments: SalTyp1 — region of the STY1607 gene, SalTyp2 — region of the STY2879 gene, SalTyp3 — region of the STBHUCCB_38510 gene of the PapD chaperone. Results. In silico analysis of LAMP primers showed that only the SalTyp 3 set has strict specificity for Salmonella enterica serovar Typhi. For the SalTyp 1 and SalTyp 2 an opportunity of false-positive reactions with some E. coli strains was shown. A LAMP method for DNA fluorescent detection of the typhoid fever causative agent was chosen. Assay has been based on a marker gene termed STBHUCCB_38510 and amplification with six specific primers. The detection limit was 20 copies/reaction in reference plasmids and the reaction time lasted for 35 minutes. The specificity of the method was tested on DNA specimens of 20 S. Typhi isolates and 90 strains of other heterologous bacteria from 24 different species. No false positive or false negative results were identified. Conclusion. The developed method can be used in clinical practice for laboratory confirmation of typhoid fever diagnosis as a part of epidemiological monitoring of environmental objects as well as food products.

The utility of trace DNA within forensic science for investigative and intelligence purposes

AbstractForensic science not only provides evidence to support criminal investigations, but also contributes to knowledge surrounding criminal phenomena. Forensic scientists are in the unique position to strategically combine information gained from traces to create intelligence that can assist the security space in keeping our communities safe. Research into the use of traces for more than just criminal investigations, with a focus on the greater “utility” of a trace, has pointed to the broader problem‐solving potential of traces when more fully exploited. Alongside this, there has continued to be an exponential increase of the use of biological traces for criminal investigations, in particular the collection of trace DNA specimens. The potential for identification, among other information, trace DNA can provide is vast, however much of the previous research has predominately focused on recovery rates which does not provide a holistic view of the value of trace DNA. This paper summarizes the current knowledge on the utility of trace DNA, as well as makes suggestions toward the creation of a decision‐making model that can inform scene of crime officers in regards to trace selection and decisions about trace analysis.This article is categorized under: Forensic Biology > DNA Databases and Biometrics Crime Scene Investigation > From Traces to Intelligence and Evidence

Plasmonic nano-bowls for monitoring intra-membrane changes in liposomes, and DNA-based nanocarriers in suspension.

Programmable nanoscale carriers, such as liposomes and DNA, are readily being explored for personalized medicine or disease prediction and diagnostics. The characterization of these nanocarriers is limited and challenging due to their complex chemical composition. Here, we demonstrate the utilization of surface-enhanced Raman spectroscopy (SERS), which provides a unique molecular fingerprint of the analytes while reducing the detection limit. In this paper, we utilize a silver coated nano-bowl shaped polydimethylsiloxane (PDMS) SERS substrate. The utilization of nano-bowl surface topology enabled the passive trapping of particles by reducing mobility, which results in reproducible SERS signal enhancement. The biological nanoparticles' dwell time in the nano-trap was in the order of minutes, thus allowing SERS spectra to remain in their natural aqueous medium without the need for drying. First, the geometry of the nano-traps was designed considering nanosized bioparticles of 50-150 nm diameter. Further, the systematic investigation of maximum SERS activity was performed using rhodamine 6 G as a probe molecule. The potential of the optimized SERS nano-bowl is shown through distinct spectral features following surface- (polyethylene glycol) and bilayer- (cholesterol) modification of empty liposomes of around 140 nm diameter. Apart from liposomes, the characterization of the highly crosslinked DNA specimens of only 60 nm in diameter was performed. The modification of DNA gel by liposome coating exhibited unique signatures for nitrogenous bases, sugar, and phosphate groups. Further, the unique sensitivity of the proposed SERS substrate displayed distinct spectral signatures for DNA micelles and drug-loaded DNA micelles, carrying valuable information to monitor drug release. In conclusion, the findings of the spectral signatures of a wide range of molecular complexes and chemical morphology of intra-membranes in their natural state highlight the possibilities of using SERS as a sensitive and instantaneous characterization alternative.

Assessment of Y chromosome copy number alterations in non-neoplastic and neoplastic leukocytes of male dogs

The loss of the Y chromosome (ChrY), also known as LOY, is a common genetic alteration observed in men. It occurs in non-neoplastic cells as an age-related change as well as in neoplastic cells of various cancer types. While well-documented in humans, LOY has not been extensively studied in non-human mammals. In this study, we developed simple digital PCR-based assays to assess the copy number of ChrY relative to the X chromosome (ChrX) and chromosome 8 (Chr8) to evaluate ChrY numerical alterations in male canine DNA specimens. Using these assays, we analyzed non-neoplastic leukocytes from 162 male dogs without hematopoietic neoplasia to investigate the occurrence of age-related LOY in non-neoplastic leukocytes. Additionally, we examined 101 tumor DNA specimens obtained from male dogs diagnosed with various types of lymphoma and leukemia to determine whether copy number alterations of the ChrY occur in canine hematopoietic cancers. Analysis of the 162 non-neoplastic leukocyte DNA specimens from male dogs of varying ages revealed a consistent ∼1:1 ChrY:ChrX ratio. This suggests that age-related LOY in non-neoplastic leukocytes is rare or absent in dogs. Conversely, a decreased or increased ChrY:ChrX ratio was detected in canine neoplastic leukocytes at varying frequencies across different canine hematopoietic malignancies (P = 0.01, Fisher's exact test). Notably, a higher incidence of LOY was observed in more aggressive cancer types. To determine if this relative LOY to ChrX was caused by changes in ChrY or ChrX, we further analyzed their relative copy numbers using Chr8 as a reference. Loss of ChrX relative to Chr8 was found in 21% (9/41) of B-cell lymphomas and 6% (1/18) of non-T-zone/high-grade T-cell lymphomas. In contrast, a subset (29%, 4/14) of T-cell chronic lymphocytic leukemia showed gain of ChrX relative to Chr8. Notably, no relative LOY to Chr8 was detected indolent hematopoietic cancers such as T-zone lymphoma (0/9) and chronic lymphocytic leukemia of B-cell (0/11) and T-cell origins (0/14). However, relative LOY to Chr8 was present in more aggressive canine hematopoietic cancers, with incidences of 24% (10/41) in B-cell lymphoma, 44% (8/18) in non-T-zone/high-grade T-cell lymphoma, and 75% (6/8) in acute leukemia. This study highlights both similarities and differences in LOY between human and canine non-neoplastic and neoplastic leukocytes. It underscores the need for further research into the role of ChrY in canine health and disease, as well as the significance of LOY across various species.

Molecular epidemiology of Enterocytozoon bieneusi from foxes and raccoon dogs in the Henan and Hebei provinces in China

BackgroundEnterocytozoon bieneusi is a zoonotic pathogen widely distributed in animals and humans. It can cause diarrhea and even death in immunocompromised hosts. Approximately 800 internal transcribed spacer (ITS) genotypes have been identified in E. bieneusi. Farmed foxes and raccoon dogs are closely associated to humans and might be the reservoir of E. bieneusi which is known to have zoonotic potential. However, there are only a few studies about E. bieneusi genotype identification and epidemiological survey in foxes and raccoon dogs in Henan and Hebei province. Thus, the present study investigated the infection rates and genotypes of E. bieneusi in farmed foxes and raccoon dogs in the Henan and Hebei provinces.ResultA total of 704 and 884 fecal specimens were collected from foxes and raccoon dogs, respectively. Nested PCR was conducted based on ITS of ribosomal RNA (rRNA), and then multilocus sequence typing (MLST) was conducted to analyze the genotypes. The result showed that infection rates of E. bieneusi in foxes and raccoon dogs were 18.32% and 5.54%, respectively. Ten E. bieneusi genotypes with zoonotic potential (NCF2, NCF3, D, EbpC, CHN-DC1, SCF2, CHN-F1, Type IV, BEB4, and BEB6) were identified in foxes and raccoon dogs. Totally 178 ITS-positive DNA specimens were identified from foxes and raccoon dogs and these specimens were then subjected to MLST analysis. In the MLST analysis, 12, 2, 7 and 8 genotypes were identified in at the mini-/ micro-satellite loci MS1, MS3, MS4 and MS7, respectively. A total of 14 multilocus genotypes were generated using ClustalX 2.1 software. Overall, the present study evaluated the infection of E. bieneusi in foxes and raccoon dogs in the Henan and Hebei province, and investigated the zoonotic potential of the E. bieneusi in foxes and raccoon dogs.ConclusionsThese findings expand the geographic distribution information of E. bieneusi’ host in China and was helpful in preventing against the infection of E. bieneusi with zoonotic potential in foxes and raccoon dogs.

Analytical Validation of a 37-Gene Next-Generation Sequencing Panel for Myeloid Malignancies and Review of Initial Findings Incorporating Updated 2022 Diagnostic and Prognostic Guidelines

Myeloid neoplasms are clonal disorders that arise via acquisition of genetic mutations leading to excessive proliferation and defective differentiation. Mutational profiling is vital as it has implications on diagnosis, prognosis, and therapeutic decision making. Next generation sequencing (NGS) has become a mainstay in the evaluation of myeloid malignancies, as it enables efficient characterisation of multiple genetic changes. Herein the analytical validation of the 37-gene Archer VariantPlex Core Myeloid panel is reported, using 58 DNA specimens with 87 single nucleotide variants (SNV) and 23 insertions/deletions (INDEL). The panel achieved good depth of coverage, 100% analytical sensitivity and specificity for SNV and INDELs ≤21bp, and 100% reproducibility, with a reportable limit of detection determined as 5%. The Archer NGS panel can accurately and reproducibly detect variants of clinical significance in myeloid neoplasms.A retrospective analysis of 535 clinical specimens tested with the Archer NGS panel showed a frequency and pattern of mutations across myeloid malignancies that were similar to other published studies. A review of the diagnostic classification of patients with acute myeloid leukaemia and myelodysplastic syndrome using the World Health Organisation 2017/2022 and International Consensus Classification 2022 guidelines, in addition to European LeukemiaNet 2017/2022 risk stratification of AML patients, was also performed to assess the utility of the molecular information provided by the Archer NGS panel.

A rapid and easy-to-use spinal muscular atrophy screening tool based on primers with high specificity and amplification efficiency for SMN1 combined with single-stranded tag hybridization assay.

Spinal muscular atrophy (SMA) is an intractable neuromuscular disorder primarily caused by homozygous deletions in exon 7 of the SMN1 gene. Early diagnosis and prompt treatment of patients with SMA have a significant impact on prognosis, and several therapies have recently been developed. Current SMA screening tests require a significant turnaround time to identify patients with suspected SMA, due both to the interval between the birth of a newborn and the collection of blood for newborn mass screening and the difficulty in distinguishing between SMN1 and SMN2, a paralog gene that requires testing in specialized laboratories. The aim of this study was therefore to develop a novel SMA screening assay that can be rapidly performed in ordinary hospitals and clinics to overcome these issues. We designed over 100 combinations of forward and reverse primers with 3' ends targeting SMN1-specific sites around exon 7, and evaluated their specificity and amplification efficiency by quantitative PCR to identify the best primer pair. Furthermore, we performed a single-stranded tag hybridization assay after PCR. To evaluate the accuracy and practicality of the newly developed assay, we analyzed saliva specimens from five patients with SMA and two SMA carriers collected in an outpatient clinic and DNA specimens from three patients with SMA and four SMA carriers from a biobank, together with those from healthy individuals. DNA and raw saliva specimens from all patients with SMA demonstrated a biallelic loss of SMN1, whereas those from carriers and healthy individuals did not. The results of 50 independent experiments were consistent for all samples. The assay could be completed within one hour. This simple and convenient new screening tool has the potential to allow patients with SMA to receive disease-modifying therapies within a shorter timeframe.

Using structural equation modeling to assess the genotoxic and mutagenic effects of heavy metal contamination in the freshwater ecosystems: A study involving Oreochromis niloticus in an urban river

Chemical pollutants represent a leading problem for aquatic ecosystems, as they can induce genetic, biochemical, and physiological changes in the species of these ecosystems, thus compromising their adaptability and survival. The Capibaribe River runs through the state of Pernambuco, located in Northeastern Brazil, and passes through areas of agricultural cultivation, densely populated cities, and industrial centers, primarily textiles. Despite its importance, few ecotoxicological studies have been conducted on its environment, and knowledge about pollution patterns and their effects on its biota is still being determined. The objective of this study was to evaluate the water quality and the damage supposed to be caused by pollutants on the DNA specimens of Nile tilapia (Oreochromis niloticus) obtained from seven strategic points of Capibaribe. Tilapia specimens and water were collected during the rainy and dry seasons from 2015 to 2017. The following characteristics were analyzed: physicochemical (six), metal concentration (seven), local pluviosity, micronuclei, and comet assay. The physicochemical and heavy metal analyses were exploratory, whereas the ecotoxicological analyses were hypothetical. To verify this hypothesis, we compared the groups of fish collected to the results of the micronuclei test and comet assay. We created a Structural Equation Model (SEM) to determine how each metal's micronuclei variables, damage index, pluviosity, and concentration were related. Our results demonstrated that the highest values for markers of genetic damage were detected at points with the highest heavy metal concentrations, especially iron, zinc, manganese, chromium, and cadmium. The SEM demonstrated that metals could explain the findings of the genotoxicity markers. Moreover, other pollutants, such as pesticides, should be considered, mainly where the river passes through rural areas. The results presented here demonstrate that the Capibaribe River has different degrees of contamination and confirm our hypothesis.

Implementing new technologies to enhance specimen quality and researcher value at the National Centralized Repository for Alzheimer’s Disease and Related Dementias

AbstractBackgroundThe National Centralized Repository for Alzheimer’s Disease and Related Dementias (NCRAD) was established in 1990 at Indiana University with the mission to support research focused on the etiology, early detection, and development of therapeutics for ADRD. NCRAD has been funded by the National Institute on Aging (NIA) since 1990 and is an integral international resource for clinical information and biological materials. As ADRD research has advanced, NCRAD and the NIA have worked together to expand the number and type of biological specimens available to the research community. NCRAD currently houses DNA, RNA, CSF, plasma, serum, stool, brain tissue, LCLs, PBMCs, fibroblasts and iPSCs from individuals with ADRD and healthy controls. NCRAD and the NIA have invested in technologies to standardize specimen processing, enhance specimen quality, and increase specimen distribution to researchers. Paired with this, all data generated from NCRAD specimens must be made available to other researchers through approved data warehouses.MethodDNA extractions are performed with standardized Chemagen chemistries on the Chemagic360 platform. Genotyping is performed with SNP Type™ assays on Juno™ and Biomark™ platforms. Automated specimen storage is executed by a Hamilton® BiOS M. Biofluid handling and sub‐aliquoting are carried out on Janus®, Biomek, and STARlet liquid‐handling platforms.ResultDNA extraction quality, consistency, and throughput increased after adopting the Chemagic360 platform. Custom SNP fingerprint genotyping of DNA specimens upon intake and distribution ensured specimen identity was not lost due to collection site errors or specimen swaps during processing. Additionally, APOE and LRRK2 G2019S genotyping has provided researchers banking specimens with valuable ADRD‐related genetic information. Automated, high‐density storage has permitted the banking of more specimens, allowing for onboarding of additional studies. Storage automation reduced the handling of specimens that could introduce warming events, contributing to confounding preanalytical variables.ConclusionRecent investments in new technologies have allowed NCRAD to accelerate ADRD research by supporting a broader range of biological specimens, increasing the number of specimens banked, limiting the effects of preanalytical variables, and shortening the time to distribute specimens. The broad availability of NCRAD specimens and the data generated from these specimens, has resulted in over 700 peer‐reviewed publications.

ACS10- Cytarabine Pharmacogenomics Score Impacts Survival in Pediatric AML Patients Treated on AAML1031 Trial and Associates with Outcome Differences in Black AML Patients

Cytarabine (ara-C) has been used to treat acute myeloid leukemia (AML) for decades. While ara-C based regimens successfully induce remission in the majority of patients, significant number of patients experience relapse. Although advances in hematopoietic stem cell transplantation (HSCT) and supportive care regimens have improved over the years, it is imperative that personalized methods to optimize pharmacological intervention and improve outcomes are needed. To identify genetic predictors of ara-C response, we recently reported an ara-C pharmacogenomics score (ACS10) consisting of 10 SNPs in nine ara-C pathway genes (PMID: 34990262). In pediatric patients treated on AML02 and AAML0531 trials, low ACS10 score (<0) was associated with poor survival as compared to patients with high score (>0). Given these promising results, we sought to investigate the ACS10 score in a large cohort of children treated on Children's Oncology Group clinical trial AAML1031 (NCT01371981). AAML1031 trial enrolled 1,097 newly diagnosed patients from 0 to 29.5 years of age that received either conventional AML treatment (ara-C, daunorubicin and etoposide (ADE): arm A) or conventional treatment with the addition of bortezomib (ADE+bortezomib: arm B). Overall, no benefit for adding bortezomib was observed (PMID: 32029509). The current study included 717 patients with both DNA specimens and clinical outcome data available. ACS10 SNPs were genotyped using Taqman assays and ACS10 score was calculated for each patient as per previous report (PMID: 34990262). Overall, for the 717 patients included in this study, 249 patients had low ACS10score (< 0) and 468 patients had a high ACS10 score (>0). When combining patients treated on arm A and arm B, ACS10 score was not associated with overall survival (OS). However, patients with low ACS10 scores had poorer event-free survival (EFS) than those with high ACS10 scores, although this difference was not statistically significant (HR: 0.827, 95% CI (0.669-1.022), P=0.078). Patients with high ACS10 scores in arm A had improved EFS (HR: 0.689, 95% CI (0.50-0.94), P=0.023, Fig1), while OS did not show significant differences (P=0.378). Excluding the patients who received HSCT, showed increased EFS for patients with high ACS10 scores as compared to low ACS10 scores when treated with standard chemotherapy (HR: 0.628, 95% CI (0.445-0.887), P=0.008 Fig 1). In contrast, a significant difference was not observed within the standard ADE+bortezomib arm (arm B). At the whole cohort level, black patients (N=84) had reduced OS (HR: 1.47, 95% CI (1.02-2.13), P=0.041) as compared to white patients, while there was no significant decrease in EFS (HR: 1.22, 95% CI (0.89-1.66), P=0.206) (N=522). Within arm A, black patients had significantly poorer outcome as compared to white patients (EFS, HR: 1.718, 95% CI (1.099-2.686), P=0.018 Fig1; OS HR: 1.80, 95% CI (1.05-3.10), P=0.032). Difference in outcome by race in arm B was not significant implying addition of bortezomib might have some benefit to black patients. Subsequent comparison by treatment arm showed a non-significant but better outcome in black patients when treated on arm B (Black patients: EFS, arm A vs. arm B: HR:0.66, 95% CI (0.37-1.17), P=0.154). Consistent with our previous investigation, low ACS10 score group was more abundant in black patients as compared to white patients (69% of black patients vs. 28% of white patients had low ACS10 score, P<0.001). These results are particularly salient given trends showing poor outcome in black as compared to white AML patients. Despite AAML1031 being one of the largest pediatric AML cohorts, the small number of black patients limited our ability to do further subset analyses by scores and arms (only 10 of 39 black patients treated on arm A had high ACS10 score). In conclusion, we demonstrate the utility of the PGx-based ACS10 score in a large cohort of newly diagnosed pediatric AML patients when treated with standard ADE induction regimen. Consistent with previous observation, black patients with low ACS10 score had inferior outcome in standard arm, however such an association was not observed for the bortezomib arm. Higher abundance of the low ACS10 SNP scores in black patients warrants further in-depth evaluation. Our future and ongoing studies include compiling data from multiple clinical trials to increase the sample size as well as expanding investigations within Hispanic/Latino AML patients

Cross-sectional study: prevalence of oedema disease Escherichia coli (EDEC) in weaned piglets in Germany at pen and farm levels

BackgroundEscherichia coli bacteria capable of producing the toxin Stx2e and possessing F18-fimbriae (edema disease E. coli, EDEC) are considered causative agents of porcine oedema disease. This disease, which usually occurs in piglets shortly after weaning, has a high lethality in affected animals and can lead to high economic losses in piglet rearing. The aim of this cross-sectional field study was to determine the prevalence of EDEC in weaned piglets in Germany at pen and farm levels.ResultsNinety-nine farms with unknown history of infections with shigatoxin-producing E. coli (STEC) and oedema disease were sampled. On each farm, up to five pens were selected for sampling (n = 481). The piglets in these pens were at an age 1–3 weeks after weaning. Single faecal samples (n = 2405) and boot swabs (n = 479) were collected from the floor. On 50 farms, cotton ropes were additionally used to collect oral fluid samples (n = 185) and rope wash out samples (n = 231) from the selected pens. All samples were analyzed by bacterial culture combined with a duplex PCR for the presence of the corresponding genes stx2e and fedA (major subunit protein of F18 fimbriae). In addition, whole DNA specimens extracted from boot swabs, oral fluid samples, and rope wash out samples were directly examined by duplex PCR for DNA of stx2e and fedA. A pen was classified as positive if at least one of the samples, regardless of the technique, yielded a positive result in the PCR, and farms were considered positive if at least one pen was classified as positive. Overall, genes stx2e and fedA were found simultaneously in 24.9% (95% CI 22.1–29.1%) of sampled pens and in 37.4% (95% CI 27.9–47.7%) of sampled farms. Regardless of the presence of F18-fimbriae, Escherichia coli encoding for Stx2e (STEC-2e) were found in 35.1% (95% CI 31.0–39.1%) of the pens and 53.5% (95% CI 44.4–63.6%) of the farms sampled.ConclusionsEscherichia coli strains considered capable to cause oedema disease in swine (EDEC) are highly prevalent in the surveyed pig producing farms in Germany. Due to intermittent shedding of EDEC and a potentially low within-farm prevalence, we recommend a combination of different sampling techniques for EDEC monitoring at pen and farm levels. Further studies are needed to understand which STEC-2e strains really pose the risk of causing severe porcine disease.

A Machine-Learning-Based Approach to Prediction of Biogeographic Ancestry within Europe.

Data obtained with the use of massive parallel sequencing (MPS) can be valuable in population genetics studies. In particular, such data harbor the potential for distinguishing samples from different populations, especially from those coming from adjacent populations of common origin. Machine learning (ML) techniques seem to be especially well suited for analyzing large datasets obtained using MPS. The Slavic populations constitute about a third of the population of Europe and inhabit a large area of the continent, while being relatively closely related in population genetics terms. In this proof-of-concept study, various ML techniques were used to classify DNA samples from Slavic and non-Slavic individuals. The primary objective of this study was to empirically evaluate the feasibility of discerning the genetic provenance of individuals of Slavic descent who exhibit genetic similarity, with the overarching goal of categorizing DNA specimens derived from diverse Slavic population representatives. Raw sequencing data were pre-processed, to obtain a 1200 character-long binary vector. A total of three classifiers were used-Random Forest, Support Vector Machine (SVM), and XGBoost. The most-promising results were obtained using SVM with a linear kernel, with 99.9% accuracy and F1-scores of 0.9846-1.000 for all classes.

B-246 The Evaluation of Actionable Mutations By Multiplex Analysis Using IntelliPlex® Lung Cancer Panel for Non-Small Cell Cancer Management

Abstract Background Understanding the genetic alterations which occur in genes associated with various cancers can improve therapy outcomes by promoting tailored treatments for each patient. Molecular profiling is therefore a crucial tool for precision medicine and enables for a personalized approach to cancer management. Current guidelines for molecular testing of Non-Small Cell Lung Cancer (NSCLC) patients involves treatment selection of a targeted Tyrosine Kinase Inhibitor based on the minimal testing of EGFR, ALK and ROS1 genes and strongly recommending the additional testing of KRAS, BRAF, MET, RET, NTRK and ERBB2 (Her2) genes to fully evaluate the patient’s mutational status. Method The IntelliPlex Lung Cancer Panel, based on PlexBio’s multiplexing πCode® technology, is an in vitro molecular amplification assay intended for the qualitative identification of 74 DNA mutations in the KRAS, NRAS, PIK3CA, BRAF, EGFR, HER2, MEK1 and AKT1 genes and 28 gene variants of the ALK, ROS1, RET, NTRK1 and MET genes. The assay is suitable for the use with formalin-fixed paraffin-embedded (FFPE) tumor tissues from NSCLC patients. The performance of IntelliPlex Lung Cancer Panel was evaluated on FFPE specimens by comparing the results with two commercially available molecular amplification assays. Discrepant samples were resolved by a corresponding digital PCR method kit. Results The IntelliPlex Lung Cancer Panel can detect DNA mutations at very low allele frequencies (as low as 0.1%) while only requiring DNA quantities of 10ng. Most RNA rearrangements are detected with 10 or more copies present in the sample while only low RNA input quantities (50 ng) are required. The IntelliPlex Lung Cancer Panel can analyze the mutational status of 102 targets in less than 5.5 h from sample to results and allows screening of up to 48 samples in parallel. Among 50 FFPE DNA specimens tested, the overall agreement for EGFR and KRAS mutations between IntelliPlex system and the comparison method were both found to be 98%. And the overall agreement after resolved the discrepancies by digital PCR was 100%. Conclusion The multiplex amplification and detection capabilities of the IntelliPlex Lung Cancer Panel provides a comprehensive and efficient way to evaluate the individual mutational status from lung cancer patients with high sensitivity and specificity. Accurate genetic analysis provides valuable information which can support precision medicine decisions for better cancer management. This can also be used further with non-invasive whole blood sampling (i.e., “liquid biopsy”) for subsequent treatment monitoring.

Effect of radiotherapy on the DNA cargo and cellular uptake mechanisms of extracellular vesicles.

In the past decades, plenty of evidence has gathered pointing to the role of extracellular vesicles (EVs) secreted by irradiated cells in the development of radiation-induced non-targeted effects. EVs are complex natural structures composed of aphospholipid bilayer which are secreted by virtually all cells and carry bioactive molecules. They can travel certain distances in the body before being taken up by recipient cells. In this review we discuss the role and fate of EVs in tumor cells and highlight the importance of DNA specimens in EVs cargo in the context of radiotherapy. The effect of EVs depends on their cargo, which reflects physiological and pathological conditions of donor cell types, but also depends on the mode of EV uptake and mechanisms involved in the route of EV internalization. While the secretion and cargo of EVs from irradiated cells has been extensively studied in recent years, their uptake is much less understood. In this review, we will focus on recent knowledge regarding the EV uptake of cancer cells and the effect of radiation in this process.

Age-specific and genotype-specific carcinogenic human papillomavirus prevalence in a country with a high cervical cancer burden: results of a cross-sectional study in Estonia

ObjectivesTo describe age-specific and type-specific carcinogenic human papillomavirus (HPV) prevalence prior to large-scale effect of HPV vaccines in Estonia and to analyse the risk factors associated with carcinogenic HPV.DesignCross-sectional study...

Angie-LAMP for diagnosis of human eosinophilic meningitis using dog as proxy: A LAMP assay for Angiostrongylus cantonensis DNA in cerebrospinal fluid

Angiostrongylus cantonensis (rat lungworm) is recognised as the leading cause of human eosinophilic meningitis, a serious condition observed when nematode larvae migrate through the CNS. Canine Neural Angiostrongyliasis (CNA) is the analogous disease in dogs. Both humans and dogs are accidental hosts, and a rapid diagnosis is warranted. A highly sensitive PCR based assay is available but often not readily accessible in many jurisdictions. An alternative DNA amplification assay that would further improve accessibility is needed. This study aimed to assess the diagnostic utility of a newly designed LAMP assay to detect DNA of globally distributed and invasive A. cantonensis and Angiostrongylus mackerrasae, the other neurotropic Angiostrongylus species, which is native to Australia. Cerebrospinal fluid (CSF) from dogs with a presumptive diagnosis of A. cantonensis infection (2020-2022) were received for confirmatory laboratory testing and processed for DNA isolation and ultrasensitive Angiostrongylus qPCR targeting AcanR3390. A newly designed LAMP assay targeting the same gene target was directly compared to the reference ultrasensitive qPCR in a diagnostic laboratory setting to determine the presence of A. cantonensis DNA to diagnose CNA. The LAMP assay (Angie-LAMP) allowed the sensitive detection of A. cantonensis DNA from archived DNA specimens (Kappa = 0.81, 95%CI 0.69-0.92; n = 93) and rapid single-step lysis of archived CSF samples (Kappa = 0.77, 95%CI 0.59-0.94; n = 52). Only A. cantonensis DNA was detected in canine CSF samples, and co-infection with A. mackerrasae using amplicon deep sequencing (ITS-2 rDNA) was not demonstrated. Both SYD.1 and AC13 haplotypes were detected using sequencing of partial cox1. The Angie-LAMP assay is a useful molecular tool for detecting Angiostrongylus DNA in canine CSF and performs comparably to a laboratory Angiostrongylus qPCR. Adaptation of single-step sample lysis improved potential applicability for diagnosis of angiostrongyliasis in a clinical setting for dogs and by extension, to humans.

THE PREFERENCE PRIORITY OF Bacillus subtilis IN UPTAKING FREE DNA DURING THE NATURAL TRANSFORMATION

Although genetic material is vertically transferred between generations via sexual or asexual reproduction, similarities in some chromosome and gene parts of unrelated organisms provide important clues for another way of transfer. The mobility of genetic information among different organisms, known as horizontal gene transfer (HGT) has immediate or delayed effects on the recipient host. One of the most notable mechanisms of HGT is natural transformation (NT), a process in which cells take free DNA from the extracellular environment and incorporate it into their chromosomes by homologous recombination. NT is widely conserved in many bacterial species as it can promote to spread of resistance genes. Although it is known that many organisms rely on HGT, there is limited information about how they decide which particular genetic material to horizontally transfer. Here, I have investigated the preference priority among different gene sources presented under certain stress conditions for Bacillus subtilis possessing NT ability. To test this, two DNA specimens (E and C) with different sequence contents of the same length were presented to B. subtilis under different stress environments (BK, BC, BE and BCE). The hypothesis was evaluated according to the analysis of the results of colonial formations on selective plates (pE, pC and pCE). The obtained data presented a strong positive correlation that the bacteria have preference priority during NT depending on a stimulator. The tendency of the bacteria to uptake useful DNA fragments in a specific environment can be suggested. For instance, the majority of colonies grow on pE plates rather than the pC and pCE when the transformation media includes erythromycin (Eryt) as an inducer. Although the data significantly overlaps with the idea claiming that the bacteria have a preference priority to uptake free DNAs during NT, further investigations are needed to support the present data and for better understanding of the phenomenon.