Initial DNA Research Articles

Evaluating Iodine-125 DNA Damage Benchmarks of Monte Carlo DNA Damage Models.

Simple SummarySimulation of initial radiation-induced DNA damage remains a major area of research, with numerous Monte Carlo models having been developed to model radiation effects on the cellular scale. While many models have been reasonably fit to a range of biological endpoints, there remains a lack of robust benchmarking data. Here, we investigate the application of a dataset on strand breaking in a single DNA strand through incorporation of radioactive Iodine-125 to distinguish between different Monte Carlo nanoscale physics models. We find that, while all models are able to effectively fit this data, they do so using significantly different best-fitting parameters and make substantially different predictions for other endpoints. These observations suggest that most nanoscale models broadly agree on the distribution of energy and can be made to fit to single datasets, but robust, multi-endpoint analysis is required to fully optimize and validate these approaches.A wide range of Monte Carlo models have been applied to predict yields of DNA damage based on nanoscale track structure calculations. While often similar on the macroscopic scale, these models frequently employ different assumptions which lead to significant differences in nanoscale dose deposition. However, the impact of these differences on key biological readouts remains unclear. A major challenge in this area is the lack of robust datasets which can be used to benchmark models, due to a lack of resolution at the base pair level required to deeply test nanoscale dose deposition. Studies investigating the distribution of strand breakage in short DNA strands following the decay of incorporated 125I offer one of the few benchmarks for model predictions on this scale. In this work, we have used TOPAS-nBio to evaluate the performance of three Geant4-DNA physics models at predicting the distribution and yield of strand breaks in this irradiation scenario. For each model, energy and OH radical distributions were simulated and used to generate predictions of strand breakage, varying energy thresholds for strand breakage and OH interaction rates to fit to the experimental data. All three models could fit well to the observed data, although the best-fitting strand break energy thresholds ranged from 29.5 to 32.5 eV, significantly higher than previous studies. However, despite well describing the resulting DNA fragment distribution, these fit models differed significantly with other endpoints, such as the total yield of breaks, which varied by 70%. Limitations in the underlying data due to inherent normalisation mean it is not possible to distinguish clearly between the models in terms of total yield. This suggests that, while these physics models can effectively fit some biological data, they may not always generalise in the same way to other endpoints, requiring caution in their extrapolation to new systems and the use of multiple different data sources for robust model benchmarking.

An external perpendicular magnetic field does not influence survival and DNA damage after proton and carbon ion irradiation in human cancer cells

Background and purposeMagnetic field effects on the radiobiological effectiveness during treatment of magnetic resonance (MRI) guided particle therapy are being debated. This study aims at assessing the influence of a perpendicular magnetic field on the biological effects in two human cancer cell lines irradiated with proton or carbon ions. Methods and materialsIn vitro cell irradiations were performed in water inside a perpendicular magnetic field of 0 and 1T for both protons and carbon ions. Samples were located in the center of a spread-out Bragg peak at 8cm water equivalent depth with a dose averaged linear energy transfer (LETd) of 4.2 or 83.4keV/μm for protons and carbon ions, respectively. Physical dose levels of 0, 0.5, 1, 2, 4 and 6Gy were employed. The irradiation field was shifted and laterally enlarged, to compensate for the beam deflection due to the magnetic field and ensure consistent and homogenous irradiations of the flasks. The human cancer cell lines SKMel (Melanoma) and SW1353 (chondrosarcoma) were selected which represent a high and a low (α/β)x ratio cell type. Cell survival curves were generated applying a linear-quadratic curve fit. DNA damage and DNA damage clearance were assessed via γH2AX foci quantification at 1 and 24h post radiation treatment. ResultsWithout a magnetic field, RBE10 values of 1.04±0.03 (SW1353) and 1.51±0.06 (SKMel) as well as RBE80 values of 0.93±0.15 (SW1353) and 2.28±0.40 (SKMel) were calculated for protons. Carbon treatments yielded RBE10 values of 1.68±0.04 (SW1353) and 2.30±0.07 (SKMel) and RBE80 values of 2.19±0.24 (SW1353) and 4.06±0.33 (SKMel). For a field strength of B=1T, RBE10 values of 1.06±0.03 (SW1353) and 1.47±0.06 (SKMel) resulted from protons, while RBE10 values of 1.70±0.05 (SW1353) and 2.37±0.08 (SKMel) were obtained for carbon ions. RBE80 values were calculated to be 1.06±0.12 (SW1353) and 2.33±0.40 (SKMel) following protons and 2.13±0.25 (SW1353) and 4.29±0.35 (SKMel) following carbon treatments. Substantially increased γH2AX foci per nucleus were found in both cell lines 1h after radiation with both ion species. At the 24h time point only carbon treated samples of both cell lines showed increased γH2AX levels. The presence of the magnetic field did neither influence the survival parameters of either cell line, nor initial DNA damage and DNA damage clearance. ConclusionsApplying a perpendicular magnetic field did not influence the cell survival, DNA repair, nor the biological effectiveness of protons or carbon ions in two human cancer cell lines.

Clinical efficiency of simultaneous CNV-seq and whole-exome sequencing for testing fetal structural anomalies

BackgroundBirth defects are responsible for approximately 7% of neonatal deaths worldwide by World Health Organization in 2004. Many methods have been utilized for examining the congenital anomalies in fetuses. This study aims to investigate the efficiency of simultaneous CNV-seq and whole-exome sequencing (WES) in the diagnosis of fetal anomaly based on a large Chinese cohort.MethodsIn this cohort study, 1800 pregnant women with singleton fetus in Hubei Province were recruited from 2018 to 2020 for prenatal ultrasonic screening. Those with fetal structural anomalies were transferred to the Maternal and Child Health Hospital of Hubei Province through a referral network in Hubei, China. After multidisciplinary consultation and decision on fetal outcome, products of conception (POC) samples were obtained. Simultaneous CNV-seq and WES was conducted to identify the fetal anomalies that can compress initial DNA and turnaround time of reports.ResultsIn total, 959 couples were finally eligible for the enrollment. A total of 227 trios were identified with a causative alteration (CNV or variant), among which 191 (84.14%) were de novo. Double diagnosis of pathogenic CNVs and variants have been identified in 10 fetuses. The diagnostic yield of multisystem anomalies was significantly higher than single system anomalies (32.28% vs. 22.36%, P = 0.0183). The diagnostic rate of fetuses with consistent intra- and extra-uterine phenotypes (172/684) was significantly higher than the rate of these with inconsistent phenotypes (17/116, P = 0.0130).ConclusionsSimultaneous CNV-seq and WES analysis contributed to fetal anomaly diagnosis and played a vital role in elucidating complex anomalies with compound causes.

DNA damage in human sperm: The sperm chromosome assay.

BackgroundSperm DNA damage is a major cause of pre‐ and post‐implantation embryonic loss in humans. However, the factors that control how and when such DNA damage occurs in human sperm are poorly understood.MethodsHere, I review information relating to sperm DNA damage that can be obtained from the sperm chromosome assays described in the existing literature.Main findingsThe sperm chromosome assays, which consist of interspecific in vitro fertilization or intracytoplasmic sperm injection using murine oocytes and subsequent chromosome analysis, indicate that the proportion of sperm showing DNA damage is initially low and there are larger numbers of sperm with potential membrane and DNA damage that are induced after ejaculation and separation from the seminal plasma. Other assays that directly detect sperm DNA (e.g., TUNEL assays, Comet assays, and acridine orange test) are not able to distinguish and detect the initial and potential DNA damage. Furthermore, the positive values in these direct assays are influenced by the frequency of immotile sperm and amorphous sperm populations.ConclusionThe findings in the sperm chromosome assays show that further improvements in sperm preparation protocols may result in the reduction of sperm DNA damage, followed by more successful outcomes in infertility treatment.

Retrospective Review and Scenario Analysis for qPCR Quality Assurance.

Quantitative polymerase chain reaction (qPCR) is a molecular method used in laboratories for detection and quantification of DNA nucleotides. Standard curves and reference values are usually combined into the PCR amplification process to calculate initial amount of target DNA molecule in a testing sample. This study is a retrospective review to examine the performance of standard curves in a qPCR-based assay for the screening of a genetic disorder in laboratories. Three different scenarios were designed to replay the historical data generated from over 3,000 qPCR assays to evaluate how omission of standard curves would affect the overall screening results. The outcomes of all the scenarios concluded that including standard curves in qPCR assays had decreased the screening specificity and accuracy, resulting in more false positives and additional retests. This was particularly true in case the initial DNA molecules were at relatively low copy numbers. It strongly suggests that the implantation of qPCR assays in diagnostic procedures should be frequently retested and reviewed. More importantly, the strategies of retrospective review and scenario analysis presented here will provide a good framework for assay validation and periodic quality assurance in laboratory.

Universally Accessible Structural Data on Macromolecular Conformation, Assembly, and Dynamics by Small Angle X-Ray Scattering for DNA Repair Insights.

Structures provide a critical breakthrough step for biological analyses, and small angle X-ray scattering (SAXS) is a powerful structural technique to study dynamic DNA repair proteins. As toxic and mutagenic repair intermediates need to be prevented from inadvertently harming the cell, DNA repair proteins often chaperone these intermediates through dynamic conformations, coordinated assemblies, and allosteric regulation. By measuring structural conformations in solution for both proteins, DNA, RNA, and their complexes, SAXS provides insight into initial DNA damage recognition, mechanisms for validation of their substrate, and pathway regulation. Here, we describe exemplary SAXS analyses of a DNA damage response protein spanning from what can be derived directly from the data to obtaining super resolution through the use of SAXS selection of atomic models. We outline strategies and tactics for practical SAXS data collection and analysis. Making these structural experiments in reach of any basic and clinical researchers who have protein, SAXS data can readily be collected at government-funded synchrotrons, typically at no cost for academic researchers. In addition to discussing how SAXS complements and enhances cryo-electron microscopy, X-ray crystallography, NMR, and computational modeling, we furthermore discuss taking advantage of recent advances in protein structure prediction in combination with SAXS analysis.

DNA condensation and formation of ultrathin nanosheets via DNA assisted self-assembly of an amphiphilic fullerene derivative.

DNA nanotechnology propose various assembly strategies to develop novel functional nanostructures utilizing unique interactions of DNA with small molecules, nanoparticles, polymers, and other biomolecules. Although, well defined nanostructures of DNA and amphiphilic small molecules were achieved through hybridization of covalently modified DNA, attaining precise organization of functional moieties through non-covalent interactions remain as a challenging task. Herein, we report mutually assisted assembly of an amphiphilic fullerene derivative and various DNA structures through non-covalent interactions, which leads to initial DNA condensation and subsequent assembly yielding ordered fullerene-DNA nanosheets. The molecular design of the cationic, amphiphilic fullerene derivative (FPy) ensures molecular solubility in the 10% DMSO-PBS buffer system and facile interactions with DNA through groove binding and electrostatic interactions of fullerene moiety and positively charged pyridinium moiety, respectively. The formation of FPy/DNA nanostructures were thoroughly investigated in the presence of λ-DNA, pBR322 plasmid DNA, and single and double stranded 20-mer oligonucleotides using UV-visible spectroscopy, AFM and TEM analysis. λ-DNA and pBR322 plasmid DNA readily condense in presence of FPy leading to micrometer sized few layer nanosheets with significant crystallinity due to ordered arrangement of fullerenes. Similarly, single and double stranded 20-mer oligonucleotides also interact efficiently with FPy and form highly crystalline nanosheets, signifying the role of electrostatic interaction and subsequent charge neutralization in the condensation triggered assembly. However, there is significant differences in the crystallinity and ordered arrangements of fullerenes between these two cases, where longer DNA form condensed structures and less ordered nanosheets while short oligonucleotides lead to more ordered and highly crystalline nanosheets, which could be attributed to the differential DNA condensation. Finally, we have demonstrated the addressability of the assembly using a cyanine modified single strand DNA, which also forms highly crystalline nanosheets and exhibit efficient quenching of the cyanine fluorescence upon self-assembly. These results open up new prospects in the development of functional DNA nanostructures through non-covalent interactions and hence have potential applications in the context of DNA nanotechnology.

A minimal motif for sequence recognition by mitochondrial transcription factor A (TFAM).

Mitochondrial transcription factor A (TFAM) plays a critical role in mitochondrial transcription initiation and mitochondrial DNA (mtDNA) packaging. Both functions require DNA binding, but in one case TFAM must recognize a specific promoter sequence, while packaging requires coating of mtDNA by association with non sequence-specific regions. The mechanisms by which TFAM achieves both sequence-specific and non sequence-specific recognition have not yet been determined. Existing crystal structures of TFAM bound to DNA allowed us to identify two guanine-specific interactions that are established between TFAM and the bound DNA. These interactions are observed when TFAM is bound to both specific promoter sequences and non-sequence specific DNA. These interactions are established with two guanine bases separated by 10 random nucleotides (GN10G). Our biochemical results demonstrate that the GN10G consensus is essential for transcriptional initiation and contributes to facilitating TFAM binding to DNA substrates. Furthermore, we report a crystal structure of TFAM in complex with a non sequence-specific sequence containing a GN10G consensus. The structure reveals a unique arrangement in which TFAM bridges two DNA substrates while maintaining the GN10G interactions. We propose that the GN10G consensus is key to facilitate the interaction of TFAM with DNA.

Generation of a single-tube quality control material for hemoglobin and DNA analyses of hemoglobinopathies.

Hemoglobinopathies are major public health problems worldwide. Accurate laboratory diagnosis of the carrier is essential, which includes initial screening, Hb analysis, and DNA analysis. For the first time, we have developed a single-tube quality control (QC) sample for these laboratory tests. The QC sample was made from a lyophilized mixture of the stabilized hemolysate with carbon monoxide saturation and the white blood cells of known thalassemia mutations. Homogeneity and stability were examined by Hb and DNA analyses on day 0 and every month for 12months, at room temperature, 4°C, and -20°C. A preliminary proficiency testing (PT) program for hemoglobinopathies using this single QC material was developed. Hemoglobin (Hb) and DNA analyses of a single-tube QC sample demonstrated satisfactory results of Hb analysis for at least five months and DNA analysis for at least one year of storage at -20°C. The results obtained from a preliminary PT program on five expert laboratories confirmed that a single tube QC sample prepared could be used as a PT item with various Hb and DNA analyses methods. A single lyophilized control sample has been generated for use in hemoglobinopathies' internal and external quality control program. Unlike other available control materials, which are used for individual testing, a single-tube QC sample generated can be used to control the pre-analytical and analytical processes of both Hb and DNA analyses and is suitable for use in the PT program of hemoglobinopathies.

Association of peripheral blood DNA methylation levels with Alzheimer’s disease progression

AbstractBackgroundIdentifying biomarkers associated with Alzheimer’s disease (AD) progression may enable patient enrichment and improve clinical trial designs. Epigenome‐wide association studies (EWAS) have revealed correlations between DNA methylation level at cytosine‐phosphate‐guanine (CpG) sites and AD pathology in the brain or diagnosis in peripheral blood.MethodWe performed cross‐sectional analysis of DNA methylation in peripheral blood as a predictor of AD progression in participants in the Alzheimer’s Disease Neuroimaging Initiative cohort.ResultThe rate of cognitive decline in participants from initial DNA sampling visit to subsequent visits was estimated by the slopes of the modified Preclinical Alzheimer Cognitive Composite (mPACC; mPACCdigit and mPACCtrailsB) and Clinical Dementia Rating Scale Sum of Boxes (CDR‐SB) plots using robust linear regression in cognitively normal (CN) participants and patients with mild cognitive impairment (MCI), respectively. Two differentially methylated positions were identified (P < 5.79 × 10−8 [Bonferroni correction threshold]): cg00386386 was associated with the slope of mPACCdigit in CN participants, and cg09422696 annotated to RP11‐661A12.5 was associated with the slope of CDR‐SB in MCI participants. No CpG sites significantly associated with diagnosis conversion status were identified. Genes involved in cognition and learning were enriched. A total of 70, 76, and 102 differentially methylated regions (DMRs) with Sidak‐corrected P < 0.05 were identified as associated with the slopes of mPACCtrailsB, mPACCdigit in CN participants, and CDR‐SB in MCI participants, respectively; these included DMRs annotated to HOXA4, HOXB6, and HOXB9. Overall, 75 and 123 DMRs were associated with conversion status in participants with CN and with MCI, respectively. The most significant DMR was annotated to an AD‐associated gene PM20D1 (chr1: 205,818,956 to 205,820,014 [13 probes], Sidak‐corrected P = 7.74 × 10−24), which was associated with both the slope of CDR‐SB and the MCI conversion status. The identified DMRs were enriched in CpG islands, promoter, and exons, including 5’ untranslated region and protein coding, whereas intergenic region, short and long interspersed nuclear element repeats, and introns were depleted.ConclusionCandidate CpG sites and regions in peripheral blood were identified as associated with the rate of cognitive decline in CN and MCI participants, respectively, in the longitudinal ADNI cohort.

Persistence of blood (DNA/RNA) on shoe soles under varying casework related conditions

Blunt force traumas by footwear can result in severe and even fatal head and upper body injuries. Oftentimes, footwear impressions are only partially available and evidential value is limited. DNA evidence on shoe soles could provide crucial evidence helping to solve crimes by linking target DNA to the activity of interest. Little is known about the persistence and detectability of biological material post such offenses and the interplay of factors affecting the analytical success. In this study, we assessed the persistence of blood on shoe soles under varying parameters such as blood location, different sneakers, weather condition, gait, amount of blood, underground and step count. We applied an optimized DNA/RNA workflow adapted to micro-traces without constraints for the primary DNA pipeline. There is a high probability to link donor DNA to the shoe sole for up to 300–400 steps, regardless of the underground, blood location, and amount of blood. Depending on the sole material and the degree of abrasion of the sole, a longer blood persistence can be observed. Considering blood, 98.2% of the initial DNA amount (1 μl initial blood volume) was lost after 100 steps walked on sole areas that are in constant contact with the ground. Proportion of foreign DNA was marginal (avg. 4.4 alleles), minimizing the probability of unintentional DNA transfer in this context. RNA typing showed high specificity but lower sensitivity than presumptive tests used for body fluid identification (BFI). Luminol is essential for targeted sampling on shoe soles, as latent blood traces (>100–200 steps) provided sufficient biological material for DNA/RNA typing. The generated data help to address the activity of interest and evaluate probabilities about prevalence of target DNA important for casework implications and assessments on activity level.

DdPCR allows 16S rRNA gene amplicon sequencing of very small DNA amounts from low-biomass samples

BackgroundOne limiting factor of short amplicon 16S rRNA gene sequencing approaches is the use of low DNA amounts in the amplicon generation step. Especially for low-biomass samples, insufficient or even commonly undetectable DNA amounts can limit or prohibit further analysis in standard protocols.ResultsUsing a newly established protocol, very low DNA input amounts were found sufficient for reliable detection of bacteria using 16S rRNA gene sequencing compared to standard protocols. The improved protocol includes an optimized amplification strategy by using a digital droplet PCR. We demonstrate how PCR products are generated even when using very low concentrated DNA, unable to be detected by using a Qubit. Importantly, the use of different 16S rRNA gene primers had a greater effect on the resulting taxonomical profiles compared to using high or very low initial DNA amounts.ConclusionOur improved protocol takes advantage of ddPCR and allows faithful amplification of very low amounts of template. With this, samples of low bacterial biomass become comparable to those with high amounts of bacteria, since the first and most biasing steps are the same. Besides, it is imperative to state DNA concentrations and volumes used and to include negative controls indicating possible shifts in taxonomical profiles. Despite this, results produced by using different primer pairs cannot be easily compared.

Performance Evaluation for Repair of HSGc-C5 Carcinoma Cell Using Geant4-DNA.

Simple SummaryTo evaluate the repair performance of HSGc-C5 carcinoma cell against radiation-induced DNA damage, a Geant4-DNA application for radiobiological research was extended by using newly measured experimental data acquired in this study. Concerning fast- and slow-DNA rejoining, the two-lesion kinetics (TLK) model parameters were adequately optimized (the repair speeds of each process were reasonably close to the DNA rejoining speed of the nonhomologous end-joining and homologous recombination pathways). The lethality probabilities of the DNA damage induced by complex double strand breaks (DSBs) and binary repair were approximately and , respectively. Using the optimized repair parameters, the Geant4-DNA simulation was able to predict the cell surviving fraction (SF) and the DNA repair kinetics.Track-structure Monte Carlo simulations are useful tools to evaluate initial DNA damage induced by irradiation. In the previous study, we have developed a Gean4-DNA-based application to estimate the cell surviving fraction of V79 cells after irradiation, bridging the gap between the initial DNA damage and the DNA rejoining kinetics by means of the two-lesion kinetics (TLK) model. However, since the DNA repair performance depends on cell line, the same model parameters cannot be used for different cell lines. Thus, we extended the Geant4-DNA application with a TLK model for the evaluation of DNA damage repair performance in HSGc-C5 carcinoma cells which are typically used for evaluating proton/carbon radiation treatment effects. For this evaluation, we also performed experimental measurements for cell surviving fractions and DNA rejoining kinetics of the HSGc-C5 cells irradiated by 70 MeV protons at the cyclotron facility at the National Institutes for Quantum and Radiological Science and Technology (QST). Concerning fast- and slow-DNA rejoining, the TLK model parameters were adequately optimized with the simulated initial DNA damage. The optimized DNA rejoining speeds were reasonably agreed with the experimental DNA rejoining speeds. Using the optimized TLK model, the Geant4-DNA simulation is now able to predict cell survival and DNA-rejoining kinetics for HSGc-C5 cells.

Geant4-DNA modeling of nanodosimetric quantities in the Jet Counter for alpha particles

The purpose of this work was to validate the calculation accuracy of nanodosimetric quantities in Geant4-DNA track structure simulation code. We implemented the Jet Counter (JC) nanodosimeter geometry in the simulation platform and quantified the impact of the Geant4-DNA physics models and JC detector performance on the ionization cluster size distributions (ICSD). ICSD parameters characterize the quality of radiation field and are supposed to be correlated to the complexity of the initial DNA damage in nanoscale and eventually the response of biological systems to radiation. We compared Monte Carlo simulations of ICSD in JC geometry performed using Geant4-DNA and PTra codes with experimental data collected for alpha particles at 3.8 MeV. We investigated the impact of simulation and experimental settings, i.e., three Geant4-DNA physics models, three sizes of a nanometer sensitive volume, gas to water density scaling procedure, JC ion extraction efficiency and the presence of passive components of the detector on the ICSD and their parameters. We found that ICSD in JC geometry obtained from Geant4-DNA simulations in water correspond well to ICSD measurements in nitrogen gas for all investigated settings, while the best agreement is for Geant4-DNA physics option 4. This work also discusses the accuracy and robustness of ICSD parameters in the context of the application of track structure simulation methods for treatment planning in particle therapy.

Identification of Complement-Related Missense Variants in Pediatric Patients with Acute and Post COVID-19 Syndromes

Background: Complement dysregulation has been documented in the molecular pathophysiology of COVID-19 and recently implicated in the relevant pediatric patient inflammatory responses.Aims: Based on our previous data in adults, we hypothesized that signatures of complement genetic variants would also be detectable in pediatric patients exhibiting COVID-19 signs and symptoms.Methods: We prospectively studied consecutive pediatric patients from our COVID-19 Units (November 2020-March 2021). COVID-19 was confirmed by reverse-transcriptase polymerase chain reaction (RT-PCR). Patient data were recorded by treating physicians that followed patients up to discharge. DNA was obtained from peripheral blood samples. Probes were designed using the Design studio (Illumina). Amplicons cover exons of complement-associated genes (C3, C5, CFB, CFD, CFH, CFHR1, CFI, CD46, CD55, MBL2, MASP1, MASP2, COLEC11, FCN1, FCN3 as well as ADAMTS13 and ΤHBD) spanning 15 bases into introns. We used 10ng of initial DNA material. Libraries were quantified using Qubit and sequenced on a MiniSeq System in a 2x150 bp run. Analysis was performed using the TruSeq Amplicon application (BaseSpace). Alignment was based on the banded Smith-Waterman algorithm in the targeted regions (specified in a manifest file). We performed variant calling with the Illumina-developed Somatic Variant Caller in germline mode and variant allele frequency higher than 20%. Both Ensembl and Refseq were used for annotation of the output files. A preliminary analysis (A) for the identification of variants of clinical significance was based on annotated ClinVar data, while a further and more selective analysis (B) was performed to identify missense complement coding variants that may biochemically contribute to the deregulation of innate responses during infection. This analysis was mainly based on the dbSNP and UniProt databases and available literature.Results: We studied 80 children and adolescents, 8 of whom developed inflammatory syndromes (MIS-C group). Among them, 41 were hospitalized and eventually all survived. 1.In our preliminary analysis, patients exhibited heterogeneous variant profiles including pathogenic, benign, likely benign, and variants of unknown significance (median number of variants: 97, range: 61-103). We found a variant of ADAMTS13 (rs2301612, missense) in 39 patients. We also detected two missense risk factor variants, previously detected in complement-related diseases: rs2230199 in C3 (33 patients); and rs800292 in CFH (36 patients). Among them, 40 patients had a combination of these characterized variants. This combination was significantly associated with the presence of dyspnea (p=0.031) and cough (p=0.042). Furthermore, 27 patients had a pathogenic variant in MBL2 (rs1800450), and 7 a pathogenic deletion in FCN3 that have been previously associated with inflammatory syndromes.2.The results of our further analysis are summarized in Figure. We identified common variants, some well represented by relatively high frequencies (>70%) (rs11098044 in CFI, rs1061170 in CFH and rs12711521 in MASP2) and others less abundant, but varying considerably between the hospitalized group, the non-admitted group and the MIS-C individuals (rs2230199 in C3, rs1065489 in CFH, rs12614 and rs641153 in CFB, rs1800450 in MBL2, rs2273346 and rs72550870 in MASP2, rs72549154 in MASP3 and rs7567833 in COLEC11, all highlighted in Figure in red).). Structurally, the majority of these common variants of interest encode charge reversal mutations. These may influence protein-protein interactions in complex formations that are important for complement activation and/or regulation.Conclusion: In pediatric COVID-19 we have detected a novel set of complement missense coding variants some of which have been implicated earlier in inflammatory syndromes and endothelial stress responses. Certain combinations of mutations of alternative and/or lectin pathway components may increase the threshold dynamics of complement consumption and therefore alter COVID-19 phenotypes. [Display omitted] DisclosuresGavriilaki: Alexion, Omeros, Sanofi Corporation: Consultancy; Gilead Corporation: Honoraria; Pfizer Corporation: Research Funding. Anagnostopoulos: Abbvie: Other: clinical trials; Sanofi: Other: clinical trials ; Ocopeptides: Other: clinical trials ; GSK: Other: clinical trials; Incyte: Other: clinical trials ; Takeda: Other: clinical trials ; Amgen: Other: clinical trials ; Janssen: Other: clinical trials; novartis: Other: clinical trials; Celgene: Other: clinical trials; Roche: Other: clinical trials; Astellas: Other: clinical trials .

SERS-PCR assays of SARS-CoV-2 target genes using Au nanoparticles-internalized Au nanodimple substrates

The reverse transcription-polymerase chain reaction (RT-PCR) method has been adopted worldwide to diagnose severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although this method has good sensitivity and specificity, there is a need to develop a more rapid diagnostic technology, given the virus’s rapid spread. However, the RT-PCR method takes a long time to diagnose SARS-CoV-2 because of the required thermocycling steps. Therefore, we developed a surface-enhanced Raman scattering (SERS)-PCR detection method using an AuNP-internalized Au nanodimple substrate (AuNDS) to shorten the diagnosis time by reducing the number of thermocycling steps needed to amplify the DNA. For the representative target markers, namely, the envelope protein (E) and RNA-dependent RNA polymerase (RdRp) genes of SARS-CoV-2, 25 RT-PCR thermocycles are required to reach a detectable threshold value, while 15 cycles are needed for magnetic bead-based SERS-PCR when the initial DNA concentration was 1.00× 105 copies/μL. However, only 8 cycles are needed for the AuNDS-based SERS-PCR. The corresponding detectable target DNA concentrations were 3.36 × 1012, 3.28 × 109, and 2.56 × 107 copies/μL, respectively. Therefore, AuNDS-based SERS-PCR is seen as being a new molecular diagnostic platform that can shorten the time required for the thermocycling steps relative to the conventional RT-PCR.

Association of peripheral blood DNA methylation level with Alzheimer\u2019s disease progression

BackgroundIdentifying biomarkers associated with Alzheimer’s disease (AD) progression may enable patient enrichment and improve clinical trial designs. Epigenome-wide association studies have revealed correlations between DNA methylation at cytosine-phosphate-guanine (CpG) sites and AD pathology and diagnosis. Here, we report relationships between peripheral blood DNA methylation profiles measured using Infinium® MethylationEPIC BeadChip and AD progression in participants from the Alzheimer’s Disease Neuroimaging Initiative (ADNI) cohort.ResultsThe rate of cognitive decline from initial DNA sampling visit to subsequent visits was estimated by the slopes of the modified Preclinical Alzheimer Cognitive Composite (mPACC; mPACCdigit and mPACCtrailsB) and Clinical Dementia Rating Scale Sum of Boxes (CDR-SB) plots using robust linear regression in cognitively normal (CN) participants and patients with mild cognitive impairment (MCI), respectively. In addition, diagnosis conversion status was assessed using a dichotomized endpoint. Two CpG sites were significantly associated with the slope of mPACC in CN participants (P < 5.79 × 10−8 [Bonferroni correction threshold]); cg00386386 was associated with the slope of mPACCdigit, and cg09422696 annotated to RP11-661A12.5 was associated with the slope of CDR-SB. No significant CpG sites associated with diagnosis conversion status were identified. Genes involved in cognition and learning were enriched. A total of 19, 13, and 5 differentially methylated regions (DMRs) associated with the slopes of mPACCtrailsB, mPACCdigit, and CDR-SB, respectively, were identified by both comb-p and DMRcate algorithms; these included DMRs annotated to HOXA4. Furthermore, 5 and 19 DMRs were associated with conversion status in CN and MCI participants, respectively. The most significant DMR was annotated to the AD-associated gene PM20D1 (chr1: 205,818,956 to 205,820,014 [13 probes], Sidak-corrected P = 7.74 × 10−24), which was associated with both the slope of CDR-SB and the MCI conversion status.ConclusionCandidate CpG sites and regions in peripheral blood were identified as associated with the rate of cognitive decline in participants in the ADNI cohort. While we did not identify a single CpG site with sufficient clinical utility to be used by itself due to the observed effect size, a biosignature composed of DNA methylation changes may have utility as a prognostic biomarker for AD progression.

What are the roles of global DNA and APC 2 gene promotor hypermethylation in multiple myeloma?

BackgroundIn today's practice, gene-based approaches come to the fore in the determination of prognosis and treatment preferences of multiple myeloma (MM). DNA methylation is one of the new approach parameters. DNA methylation occurs by the addition of a methyl group to cytosines in CpG dinucleotides. In this study, besides comparing the global DNA and APC 2 gene promotor hypermethylation between our patients with MM and healthy control group, we aimed to demonstrate the effect of hypermethylation on MM treatment responses and survival.Methods and results38 patients diagnosed with MM between January 2016 and January 2020 and 50 healthy controls were included in the study. The initial hypermethylation of the patients and the healthy control group were statistically analyzed. In addition, the increase in hypermethylation in the MM group before and after the first series of treatments were analyzed within themselves. There is a significant difference between the patients with MM diagnosis and the healthy control group in terms of the initial global hypermethylation (P = 0.001). In patients with MM, hypermethylation was significantly higher. Global hypermethylation in the post-treatment measurements was significantly increased in comparison to the pre-treatment state (P = 0.012). In terms of APC 2 promotor gene-specific hypermethylation, no significant differences were detected between pre- and post-treatment values (P = 0.368).ConclusionsThis study represents valuable data with the initial global DNA hypermethylation results in the MM patient group and the increase in hypermethylation post-treatment. it will shed light on future studies.

Evaluating Mixture Solution™- rapid and non-MCMC probabilistic mixture analysis.

We compare DNA mixture analysis via DNAˑVIEW® Mixture Solution™ and the current combined probability of inclusion (CPI) method of the South African Police Service (SAPS). South Africa has a high incidence of property-related crimes and sexual offences and consequently a great deal of low-template (LT-DNA) forensic DNA mixture casework and a perpetual backlog. A range of casework and laboratory-prepared sexual assault mixtures with initial male DNA amounts varying from about 2 to 200 cells were analysed to evaluate the benefits of a continuous model program. Unfortunately CPI methods are nearly useless for LT-DNA cases because of dropout-common from a mixture contribution of fewer than 20 or 30 cells. We further argue that proposed CPI elaborations to circumvent dropout lack supporting research or even explanation. Mixture Solution models mixture data as continuous rather than binary, with a mathematically coherent ("probabilistic") model which incorporates dropout and other phenomena realistically. Much more information is thereby utilised resulting in applicability to more cases (7 or fewer contributor cells suffice), stronger evidence against a suspect who is a mixture contributor and stronger evidence to absolve a non-contributor. Mixture Solution incidentally provides information which, along with rfu data, allows estimating contributions in terms of number of cells, which is a useful perspective. The method of calculation is explained.

Common Kinetic Mechanism of Abasic Site Recognition by Structurally Different Apurinic/Apyrimidinic Endonucleases.

Apurinic/apyrimidinic (AP) endonucleases Nfo (Escherichia coli) and APE1 (human) represent two conserved structural families of enzymes that cleave AP-site–containing DNA in base excision repair. Nfo and APE1 have completely different structures of the DNA-binding site, catalytically active amino acid residues and catalytic metal ions. Nonetheless, both enzymes induce DNA bending, AP-site backbone eversion into the active-site pocket and extrusion of the nucleotide located opposite the damage. All these stages may depend on local stability of the DNA duplex near the lesion. Here, we analysed effects of natural nucleotides located opposite a lesion on catalytic-complex formation stages and DNA cleavage efficacy. Several model DNA substrates that contain an AP-site analogue [F-site, i.e., (2R,3S)-2-(hydroxymethyl)-3-hydroxytetrahydrofuran] opposite G, A, T or C were used to monitor real-time conformational changes of the tested enzymes during interaction with DNA using changes in the enzymes’ intrinsic fluorescence intensity mainly caused by Trp fluorescence. The extrusion of the nucleotide located opposite F-site was recorded via fluorescence intensity changes of two base analogues. The catalytic rate constant slightly depended on the opposite-nucleotide nature. Thus, structurally different AP endonucleases Nfo and APE1 utilise a common strategy of damage recognition controlled by enzyme conformational transitions after initial DNA binding.