Prediction of Transcription Factor Binding Sites on Cell-Free DNA Based on Deep Learning.

Transcription factors (TFs) are important regulatory elements for vital cellular activities, and the identification of transcription factor binding sites (TFBS) can help to explore gene regulatory mechanisms. Research studies have proved that cfDNA (cell-free DNA) shows relatively higher coverage at TFBS due to the protection by TF from degradation by nucleases and short fragments of cfDNA are enriched in TFBS. However, there are still great difficulties in the noninvasive identification of TFBSs from experimental techniques. In this study, we propose a deep learning-based approach that can noninvasively predict TFBSs of cfDNA by learning sequence information from known TFBSs through convolutional neural networks. Under the addition of long short-term memory, our model achieved an area under the curve of 84%. Based on this model to predict cfDNA, we found consistent motifs in cfDNA fragments and lower coverage occurred upstream and downstream of these cfDNA fragments, which is consistent with a previous study. We also found that the binding sites of the same TF differ in different cell lines. TF-specific target genes were detected from cfDNA and were enriched in cancer-related pathways. In summary, our method of locating TFBSs from plasma has the potential to reflect the intrinsic regulatory mechanism from a noninvasive perspective and provide technical guidance for dynamic monitoring of disease in clinical practice.

Surpassing sensitivity limits in liquid biopsy.

Attenuation of cell-free DNA clearance in vivo is an alternative strategy to maximize recovery.

A novel circulating biomarker lnc-MALAT1 for acute myocardial infarction: Its relationship with disease risk, features, cytokines, and major adverse cardiovascular events.

OBJECTIVE: Long noncoding RNA MALAT1 (lnc-MALAT1) modulates atherosclerotic progression, myocardial ischemia injury, and systematic inflammation, which may be closely involved in acute myocardial infarction (AMI) pathogenesis. Thus, the current study intended to explore the relationship of lnc-MALAT1 to disease risk, features, cytokines, and prognostication in AMI patients. METHODS: This multicenter study consecutively enrolled 160 newly diagnosed AMI patients and 50 controls (angina pectoris patients). Their peripheral blood mononuclear cells were obtained to measure lnc-MALAT1 by RT-qPCR. Serum cytokines in AMI patients were detected by ELISA. In addition, AMI patients were followed up for major adverse cardiovascular event (MACE) risk evaluation. RESULTS: Lnc-MALAT1 was higher in AMI patients than in controls (median: 2.245 vs. 0.996, p = 0.004), and it also presented a good capacity for differentiating AMI patients from controls with an area under the curve of 0.823. Lnc-MALAT1 was positively related to C-reactive protein (p = 0.005), low-density lipoprotein cholesterol (p = 0.022), cardiac troponin I (p = 0.021), and infarct size (p = 0.007), but not other biochemical indexes in AMI patients. Meanwhile, lnc-MALAT1 was positively associated with tumor necrosis factor-alpha (p = 0.001), interleukin (IL)-6 (p = 0.031), IL-17A (p = 0.042), vascular cell adhesion molecule-1 (p = 0.004), and intercellular adhesion molecule-1 (p = 0.021) among AMI patients. Importantly, after categorization, lnc-MALAT1 high (vs. low) was related to an elevated MACE accumulation rate (p = 0.035); furthermore, a higher lnc-MALAT1 quartile showed a trend to be linked with an increased MACE accumulation rate (p = 0.092). CONCLUSION: Lnc-MALAT1 may serve as a biomarker for AMI risk, infarct size, inflammation and prognosis, but further validation by large-scale studies is needed.

Diagnostic utility of pleural cell-free nucleic acids in undiagnosed pleural effusions.

Pleural effusion (PE) is a common sign caused by various disorders. Microbiology, histology and cytology are reference standards for these disorders. However, these diagnostic tools have limitations, including invasiveness, high cost, long turnaround time, and observer-dependent. Soluble biomarkers in pleural fluid (PF) are promising diagnostic tools because they are mininvasive, economical, and objective. Recent studies have revealed that some cell-free nucleic acids (e.g., DNA, mRNA, microRNA, and lncRNA) in PF are potential diagnostic markers for many disorders. Here, we review the performance of PF cell-free nucleic acids for differentiating and stratification of PE.

Analysis of cf-mtDNA and cf-nDNA fragment size distribution using different isolation methods in BV-2 cell supernatant of starvation-induced autophagy.

Fragment size distribution, the important biological properties of cell-free DNA (cfDNA), provides useful information required for diagnostic assay development. However, besides methodological discrepancies, it varies due to the complicated origins and occurrences of in vivo cfDNA. In addition, limited data are available concerning the cfDNA associated with autophagy and distributional difference between cf-mitochondrial DNA (cf-mtDNA) and cf-nuclear DNA (cf-nDNA) fragments. Here we developed an in vitro model of mouse microglial cell (BV-2) with starvation-induced autophagy, in which cfDNA was isolated from the cell supernatant by ultrafiltration (UF) and column-based commercial kit (CC), respectively. Using Agilent 2100 Bioanalyzer, a DNA ladder pattern as the presence of peaks corresponding to mono-, di- and tri-nucleosomes was clearly visualized both in isolation products of UF and CC. However, we also detected shorter fragments than mono-nucleosome by UF. In comparing the UF and CC, we found that the former produced the higher recovery efficiency for spiked-in DNA of shorter fragments than mono-nucleosome in both water and medium, but the latter was superior for spiked-in DNA fragments which were longer than or equal to mono-nucleosome in medium. Combined with these two isolation methods, we have observed that autophagy-associated cf-mtDNA and cf-nDNA were both highly enriched in

Postsynthetic Modification of the Magnetic Zirconium-Organic Framework for Efficient and Rapid Solid-Phase Extraction of DNA.

In recent years, several approaches have been applied to modify metal-organic frameworks (MOFs) owing to their excellent structural tunability such as higher extraction efficiency than that of primitive crystals. Herein, Zr-based MOFs (UiO-66-NH2) with a suitable size modulated by acetic acid were successfully synthesized for effective DNA extraction. The bonding conformations and adsorption mechanism indicated a high affinity between UiO-66-NH2 and the DNA molecules. Furthermore, Fe3O4 nanoparticles were immobilized on the UiO-66-NH2 surface to allow MOFs with magnetism. The magnetic zirconium-organic framework (MZMOF) retained the intact structure of MOFs and simplified subsequent extraction operations. In the DNA recovery investigation, MZMOF showed high recovery efficiency for both short-stranded DNA (90.4%) and pseudovirus DNA (95.1%). In addition, it showed superior DNA extraction efficiency from plasma (57.6%) and swab preservation solution (86.5%). The prepared MZMOF was employed for highly specific extraction of viral DNA and cfDNA from samples. To further simplify the extraction process, MZMOF was applied to immiscible phase filtration assisted by a surface tension (IFAST) chip for facilitating rapid DNA extraction with sensitive point-of-care testing. The developed MZMOF-based extraction method has significant potential for increasing the demand for rapid and efficient nucleic acid extraction.

Sequencing Cell-free Fetal DNA in Pregnant Women With GCK-MODY.

CONTEXT: Individuals with monogenic diabetes due to inactivating glucokinase (GCK) variants typically do not require treatment, except potentially during pregnancy. In pregnancy, fetal GCK genotype determines whether treatment is indicated, but noninvasive methods are not clinically available. OBJECTIVE: This work aims to develop a method to determine fetal GCK genotype noninvasively using maternal cell-free fetal DNA. METHODS: This was a proof-of-concept study involving 3 pregnant women with a causal GCK variant that used information from 1) massive parallel sequencing of maternal plasma cell-free DNA, 2) direct haplotype sequences of maternal genomic DNA, and 3) the paternal genotypes to estimate relative haplotype dosage of the pathogenic variant-linked haplotype. Statistical testing of variant inheritance was performed using a sequential probability ratio test (SPRT). RESULTS: In each of the 3 cases, plasma cell-free DNA was extracted once between gestational weeks 24 and 36. The fetal fraction of cell-free DNA ranged from 21.8% to 23.0%. Paternal homozygous alleles that were identical to the maternal GCK variant-linked allele were not overrepresented in the cell-free DNA. Paternal homozygous alleles that were identical to the maternal wild-type-linked allele were significantly overrepresented. Based on the SPRT, we predicted that all 3 cases did not inherit the GCK variant. Postnatal infant genotyping confirmed our prediction in each case. CONCLUSION: We have successfully implemented a noninvasive method to predict fetal GCK genotype using cell-free DNA in 3 pregnant women carrying an inactivating GCK variant. This method could guide tailoring of hyperglycemia treatment in pregnancies of women with GCK monogenic diabetes.

Longer DNA exhibits greater potential for cell-free gene expression.

Cell-free gene expression systems have been valuable tools for understanding how transcription/translation can be regulated in living cells. Many studies have investigated the determining factors that affect gene expression. Here we report the effect of the length of linearized reporter DNAs encoding the firefly luciferase gene so as to exclude the influence of supercoiling. It is found that longer DNA molecules exhibit significantly greater potency in gene expression; for example, the expression level for DNA with 25.7 kbp is 1000-times higher than that for DNA of 1.7 kbp. AFM observation of the DNA conformation indicates that longer DNA takes shrunken conformation with a higher segment density in the reaction mixture for gene expression, in contrast to the stiff conformation of shorter DNA. We propose an underlying mechanism for the favorable effect of longer DNA on gene expression in terms of the enhancement of access of RNA polymerase to the shrunken conformation. It is expected that the enhancement of gene expression efficiency with a shrunken DNA conformation would also be a rather general mechanism in living cellular environments.

Length-Dependent, Single-Molecule Analysis of Short Double-Stranded DNA Fragments through Hydrogel-Filled Nanopores: A Potential Tool for Size Profiling Cell-Free DNA.

Fast sampling followed by sequence-independent sensing and length-dependent detection of short double-stranded DNA fragments, the size of those found in blood and other bodily fluids, is achieved using engineered molecular sensors, dubbed hydrogel-filled nanopores (HFNs). Fragments as short as 100 base pairs were blindly sampled and concentrated at the tip of an HFN before reversing the applied potential to detect and distinguish individual molecules based on fragment length as they translocate out of the nanopore. A remarkable 16-fold increase in the signal-to-noise ratio was observed in the eject configuration compared to the load configuration, enabling the resolution of fragments with a size difference of 50 nucleotides in length. This fast and versatile technology offers great tunability for both sampling and detection. While increasing sampling time leads to an increase in the local DNA concentration at the tip prior to detection, a linear correlation between the peak current and DNA fragment size enables good resolution of fragments up to 250 bp long.

Characterization of fragment sizes, copy number aberrations and 4-mer end motifs in cell-free DNA of hepatocellular carcinoma for enhanced liquid biopsy-based cancer detection.

Circulating cell-free DNA (cfDNA) fragmentomics, which encompasses the measurement of cfDNA length and short nucleotide motifs at the ends of cfDNA molecules, is an emerging field for cancer diagnosis. The utilization of cfDNA fragmentomics for the diagnosis of patients with hepatocellular carcinoma (HCC) caused by hepatitis B virus (HBV) is currently limited. In this study, we utilized whole-genome sequencing data of cfDNA in samples from patients with HCC (n = 197) and HBV (n = 187) to analyze the association of fragment size selection (< 150 bp) with tumor fraction (TF), copy number variation (CNV) alterations and the change in the proportion of 4-mer end motifs in HCC and HBV samples. Our analyses identified five typical CNV markers (i.e. loss in chr1p, chr4q and chr8p, and gain in chr1q and chr8q) in cfDNA with a cumulatively positive rate of ˜ 95% in HCC samples. Size selection (< 150 bp) significantly enhanced TF and CNV signals in HCC samples. Additionally, three 4-mer end motifs (CCCA, CCTG and CCAG) were identified as preferred end motifs in HCC samples. We identified 139 end motifs significantly associated with fragment size that showed similar patterns of associations between patients with HCC and HBV, suggesting that end motifs might be inherently coupled with fragment size by a ubiquitous mechanism. Here we conclude that CNV markers, fragment size selection and end-motif pattern in cfDNA have potential for effective detection of patients with HCC.

High-Efficiency Protection of Linear DNA in Cell-Free Extracts from Escherichia coli and Vibrio natriegens.

The field of cell-free synthetic biology is an emerging branch of engineered biology that allows for rapid prototyping of biological designs and, in its own right, is becoming a venue for the in vitro operation of gene circuit-based sensors and biomanufacturing. To date, the related DNA encoded tools that operate in cell-free reactions have primarily relied on plasmid DNA inputs, as linear templates are highly susceptible to degradation by exonucleases present in cell-free extracts. This incompatibility has precluded significant throughput, time and cost benefits that could be gained with the use of linear DNA in the cell-free expression workflow. Here to tackle this limitation, we report that terminal incorporation of Ter binding sites for the DNA-binding protein Tus enables highly efficient protection of linear expression templates encoding mCherry and deGFP. In Escherichia coli extracts, our method compares favorably with the previously reported GamS-mediated protection scheme. Importantly, we extend the Tus-Ter system to Vibrio natriegens extracts, and demonstrate that this simple and easily implemented method can enable an unprecedented plasmid-level expression from linear templates in this emerging chassis organism.

Alterations of 5-hydroxymethylcytosines in circulating cell-free DNA reflect retinopathy in type 2 diabetes.

Diabetic retinopathy (DR) is a common microvascular complication that may cause severe visual impairment and blindness in patients with type 2 diabetes mellitus (T2DM). Early detection of DR will expand the range of potential treatment options and enable better control of disease progression. Epigenetic dysregulation has been implicated in the pathogenesis of microvascular complications in patients with T2DM. We sought to explore the diagnostic value of 5-hydroxymethylcytosines (5hmC) in circulating cell-free DNA (cfDNA) for DR, taking advantage of a highly sensitive technique, the 5hmC-Seal. The genome-wide 5hmC profiles in cfDNA samples from 35 patients diagnosed with DR and 35 age-, gender-, diabetic duration-matched T2DM controls were obtained using the 5hmC-Seal, followed by a case-control analysis and external validation. The genomic distribution of 5hmC in cfDNA from patients with DR reflected potential gene regulatory relevance, showing co-localization with histone modification marks for active expression (e.g., H3K4me1). A three-gene signature (MESP1, LY6G6D, LINC01556) associated with DR was detected using the elastic net regularization on the multivariable logistic regression model, showing high accuracy to distinguish patients with DR from T2DM controls (AUC [area under curve] = 91.4%; 95% CI [confidence interval], 84.3- 98.5%), achieving a sensitivity of 88.6% and a specificity of 91.4%. In an external testing set, the 5hmC model detected 5 out of 6 DR patients and predicted 7 out of 8 non-DR patients with other microvascular complications. Circulating cfDNA from patients with DR contained 5hmC information that could be exploited for DR detection. As a novel non-invasive approach, the 5hmC-Seal holds the promise to be an integrated part of patient care and surveillance tool for T2DM patients.

Electron Acceptors Co-Regulated Self-Powered Photoelectrochemical Strategy and Its Application for Circulating Tumor Nucleic Acid Detection Coupled with Recombinase Polymerase Amplification.

Biosensor working in a self-powered mode has been widely concerned because it produces a signal when the bias potential is 0 V. However, the self-powered mode is used only when the materials have self-powered properties. Conversion of non-self-powered to self-powered through molecular regulation can solve this problem effectively. Here, we fabricated a self-powered photoelectrochemical mode based on co-regulation of electron acceptors methylene blue (MB) and p-nitrophenol (p-NP). AuNPs@ZnSe nanosheet-modified gold electrode (AuNPs@ZnSeNSs/GE) gave a small photocurrent at 0 V. In the presence of MB and p-NP, AuNPs@ZnSeNSs/GE gave the strongest photocurrent at 0 V. Accordingly, an electron acceptor co-regulated self-powered photoelectrochemical assay was fabricated. As proof-of-concept demonstrations, this assay was applied for prostate cancer circulating tumor nucleic acid biomarker, KLK2 and PCA3, detection combined with in situ recombinase polymerase amplification strategy. This assay generated a strong photocurrent and was sensitive to the variation of KLK2 and PCA3 concentration. The limits of detection were 30 and 32 aM, respectively. We anticipate this electron acceptor co-regulated self-powered photoelectrochemical mode to pave a new way for the development of self-powered sensing.

The biomolecule corona of lipid nanoparticles contains circulating cell-free DNA.

The spontaneous adsorption of biomolecules onto the surface of nanoparticles (NPs) in complex physiological biofluids has been widely investigated over the last decade. Characterisation of the protein composition of the 'biomolecule corona' has dominated research efforts, whereas other classes of biomolecules, such as nucleic acids, have received no interest. Scarce, speculative statements exist in the literature about the presence of nucleic acids in the biomolecule corona, with no previous studies attempting to describe the contribution of genomic content to the blood-derived NP corona. Herein, we provide the first experimental evidence of the interaction of circulating cell-free DNA (cfDNA) with lipid-based NPs upon their incubation with human plasma samples, obtained from healthy volunteers and ovarian carcinoma patients. Our results also demonstrate an increased amount of detectable cfDNA in patients with cancer. Proteomic analysis of the same biomolecule coronas revealed the presence of histone proteins, suggesting an indirect, nucleosome-mediated NP-cfDNA interaction. The finding of cfDNA as part of the NP corona, offers a previously unreported new scope regarding the chemical composition of the 'biomolecule corona' and opens up new possibilities for the potential exploitation of the biomolecule corona for the enrichment and analysis of blood-circulating nucleic acids.

Detection of cell-free DNA nanoparticles in insulator based dielectrophoresis systems.

In this paper, a semi-analytical investigation was performed to study the effect of the geometrical parameters of insulator-based dielectrophoresis (iDEP) systems for cell free DNA (cfDNA) trapping. For this purpose, first electrical potential and fluid flow fields were calculated by solving the governing equations including Poisson and Navier-stokes equations with appropriate boundary conditions (BCs) and then a Lagrangian approach was utilized to analyze the motion of cfDNA under the most important forces affected on it including Brownian, Drag, electrophoresis and dielectrophoresis (DEP) forces. The effect of the different parameters such as the electrical conductivity of the medium, shape and geometrical parameters of the insulators on the dielectrophoretic behavior of cfDNA was studied and the optimal value of these parameters was presented. Finally, in order to predict the minimum voltage required for cfDNA trapping, artificial neural network (ANN) was utilized and a relation between input and output parameters was introduced.

Deoxyribonucleases and Their Applications in Biomedicine.

Extracellular DNA, also called cell-free DNA, released from dying cells or activated immune cells can be recognized by the immune system as a danger signal causing or enhancing inflammation. The cleavage of extracellular DNA is crucial for limiting the inflammatory response and maintaining homeostasis. Deoxyribonucleases (DNases) as enzymes that degrade DNA are hypothesized to play a key role in this process as a determinant of the variable concentration of extracellular DNA. DNases are divided into two families-DNase I and DNase II, according to their biochemical and biological properties as well as the tissue-specific production. Studies have shown that low DNase activity is both, a biomarker and a pathogenic factor in systemic lupus erythematosus. Interventional experiments proved that administration of exogenous DNase has beneficial effects in inflammatory diseases. Recombinant human DNase reduces mucus viscosity in lungs and is used for the treatment of patients with cystic fibrosis. This review summarizes the currently available published data about DNases, their activity as a potential biomarker and methods used for their assessment. An overview of the experiments with systemic administration of DNase is also included. Whether low-plasma DNase activity is involved in the etiopathogenesis of diseases remains unknown and needs to be elucidated.

Plasma RNA sequencing of extracellular RNAs reveals potential biomarkers for non-small cell lung cancer.

BACKGROUND: Lung cancer is one of the most common malignancies, and it has extremely high incidence and mortality rates. Although there have been many studies focused on lung cancer biomarkers, few have reported the extracellular RNA profiles of lung cancer. In this study, we used RNA-seq technology to analyze extracellular RNAs in low volume peripheral blood plasma; we compared the differentially expressed genes from the plasma of non-small cell lung cancer (NSCLC) patients with that of healthy controls. METHODS: We used RNA-seq technology and bioinformatics to analyze the extracellular RNA (exRNA) sequences of 12 human plasma samples (500 µl per sample), 6 from NSCLC patients and 6 from healthy controls. Subsequently, we used gene ontology (GO) enrichment, KEGG analysis and coexpression experiments to compare the differentially expressed genes (DEGs) and identify tumor biomarkers that were highly correlated with NSCLC. These DEGs were further verified by quantitative PCR. RESULTS: Approximately 20 million clean reads were produced for each plasma sample; 50-80% of the reads aligned to the human references, and hundreds of thousands of reads were counted in each plasma sample. In addition, a total of 640 genes (368 upregulated and 272 downregulated) were differentially expressed between NSCLC plasma and normal plasma. Further, we identified 7 key DEGs that are highly correlated with lung tumorigenesis: COX1, COX2, COX3, ND1, ND2, ND4L, and ATP6. CONCLUSION: exRNA-seq from a small amount (400-500 µl) of plasma opens new possibilities for exploring lung cancer biomarkers in the plasma.

The Detection of 8-Oxo-7,8-Dihydro-2'-Deoxyguanosine in Circulating Cell-Free DNA: A Step Towards Longitudinal Monitoring of Health.

Chronic stress appears to accelerate biological aging, and oxidative damage is an important potential mediator of this process. Many chronic diseases are accompanied by an increase in overall oxidation of genomic DNA. In course of exposure to daily environmental insults, DNA accumulates oxidative damage, which is, in part, repaired, while the cells with the most damaged DNA die either by necrosis or by apoptosis. The oxidized DNA released from the dying cells contributes to the pool of cell-free/extracellular DNA present in plasma and other biological fluids. This cell-free DNA contains a great deal of 8-oxodG bases. The ratio of 8-oxo-dG and unmodified guanine may serve as a cumulative biomarker of stress encountered by a human body within a previous 24 h-period. This true end-point biomarker may outperform other short-lived molecules that reflect only the most current state of oxidant stress. Patient-specific baselines for oxidative damage may be established by measuring of 8-oxo-dG in circulating DNA. Longitudinal profiling of oxiDNA may aid in reliable quantification of the effects of various self-administered nutraceutical and lifestyle based health interventions. Development of wearable electrochemical sensor patches that will quantify oxiDNA in near real-time is warranted to produce life- and health-modifying event awareness feedback.

[Analysis of DNA methylation alterations in cellfree DNA fraction during colorectal cancer development]. / A vastagbéldaganatok kialakulásában szerepet játszó DNS-metilációs eltérések vizsgálata a sejten kívüli DNS-frakcióban.

During colorectal cancer (CRC) development, in addition to genetic alterations, several epigenetic changes, including DNA methylation in the promoter regions accumulate in tumor cells. Cell-free DNA (cfDNA) in the circulatory system can originate also from tumor tissue; therefore the evaluation of methylated cfDNA in the plasma can be a promising method for early cancer screening. In my Ph.D., I have investigated the rate of cfDNA's release and stability using animal models. I aimed to compile an epigenetic marker panel, which contains genes with altered DNA methylation patterns in the healthy-colorectal adenoma-cancer sequence. I have found that the methylation level of SFRP1, SFRP2, SDC2, and PRIMA1 gene promoters has already increased in adenoma stages in both tissue and plasma samples. Immunohistochemistry analyses indicated decreasing protein expression in parallel with elevated methylation. According to our results, cfDNA amount and the methylation have been influenced by DNA isolation and blood collection methods.

The fragmentation patterns of maternal plasma cell-free DNA and its applications in non-invasive prenatal testing.

The discovery of cell-free DNA (cfDNA) in maternal plasma has opened up new promises for the development of non-invasive prenatal testing (NIPT). Application of cfDNA in NIPT of fetus diseases and abnormalities is restricted by the low amount of fetal DNA molecules in maternal plasma. Fetus-derived cfDNA in maternal plasma are shorter than maternal DNA, thus leveraging the maternal and fetus-derived cfDNA molecules size difference has become a novel and more accurate method for NIPT. However, multiple biological properties such as size distribution of plasma DNA, proportion of fetal-derived DNA and methylation levels in maternal plasma across different gestational ages still remain largely unknown. Further insights into the size distribution and fragmentation pattern of circulating plasma cfDNA will shed light on the origin and fragmentation mechanisms of cfDNA during physiological and pathological processes in prenatal diseases and enhance our ability to take the advantage of plasma cfDNA as a molecular diagnostic tool. In the review, we start by summarizing the research techniques for the determination of the fragmentation profiles of cfDNA in maternal plasma. We then summarize the main progress and findings in size profiles of maternal plasma cfDNA and cffDNA. Finally, we discuss the potential diagnostic applications of plasma cfDNA size profiling.