Cell-free DNA (cfDNA) in human plasma is a class of biomarkers with many current and potential future diagnostic applications. Recent studies have shown that cfDNA molecules are not randomly ...fragmented and possess information related to their tissues of origin. Pathologies causing death of cells from particular tissues result in perturbations in the relative distribution of DNA from the affected tissues. Such tissue-of-origin analysis is particularly useful in the development of liquid biopsies for cancer. It is therefore of value to accurately determine the relative contributions of the tissues to the plasma DNA pool in a simultaneous manner. In this work, we report that in open chromatin regions, cfDNA molecules show characteristic fragmentation patterns reflected by sequencing coverage imbalance and differentially phased fragment end signals. The latter refers to differences in the read densities of sequences corresponding to the orientation of the upstream and downstream ends of cfDNA molecules in relation to the reference genome. Such cfDNA fragmentation patterns preferentially occur in tissue-specific open chromatin regions where the corresponding tissues contributed DNA into the plasma. Quantitative analyses of such signals allow measurement of the relative contributions of various tissues toward the plasma DNA pool. These findings were validated by plasma DNA sequencing data obtained from pregnant women, organ transplantation recipients, and cancer patients. Orientation-aware plasma DNA fragmentation analysis therefore has potential diagnostic applications in noninvasive prenatal testing, organ transplantation monitoring, and cancer liquid biopsy.
Abstract
Background
Double-stranded DNA in plasma is known to carry single-stranded ends, called jagged ends. Plasma DNA jagged ends are biomarkers for pathophysiologic states such as pregnancy and ...cancer. It remains unknown whether urinary cell-free DNA (cfDNA) molecules have jagged ends.
Methods
Jagged ends of cfDNA were detected by incorporating unmethylated cytosines during a DNA end-repair process, followed by bisulfite sequencing. Incorporation of unmethylated cytosines during the repair of the jagged ends lowered the apparent methylation levels measured by bisulfite sequencing and were used to calculate a jagged end index. This approach is called jagged end analysis by sequencing.
Results
The jagged end index of urinary cfDNA was higher than that of plasma DNA. The jagged end index profile of plasma DNA displayed several strongly oscillating major peaks at intervals of approximately 165 bp (i.e., nucleosome size) and weakly oscillating minor peaks with periodicities of approximately 10 bp. In contrast, the urinary DNA jagged end index profile showed weakly oscillating major peaks but strongly oscillating minor peaks. The jagged end index was generally higher in nucleosomal linker DNA regions. Patients with bladder cancer (n = 46) had lower jagged end indexed of urinary DNA than participants without bladder cancer (n = 39). The area under the curve for differentiating between patients with and without bladder cancer was 0.83.
Conclusions
Jagged ends represent a property of urinary cfDNA. The generation of jagged ends might be related to nucleosomal structures, with enrichment in linker DNA regions. Jagged ends of urinary DNA could potentially serve as a new biomarker for bladder cancer detection.
Circulating tumor-derived cell-free DNA (ctDNA) analysis offers an attractive noninvasive means for detection and monitoring of cancers. Evidence for the presence of cancer is dependent on the ...ability to detect features in the peripheral circulation that are deemed as cancer-associated. We explored approaches to improve the chance of detecting the presence of cancer based on sequence information present on ctDNA molecules. We developed an approach to detect the total pool of somatic mutations. We then investigated if there existed a class of ctDNA signature in the form of preferred plasma DNA end coordinates. Cell-free DNA fragmentation is a nonrandom process. Using plasma samples obtained from liver transplant recipients, we showed that liver contributed cell-free DNA molecules ended more frequently at certain genomic coordinates than the nonliver-derived molecules. The abundance of plasma DNA molecules with these liver-associated ends correlated with the liver DNA fractions in the plasma samples. Studying the DNA end characteristics in plasma of patients with hepatocellular carcinoma and chronic hepatitis B, we showed that there were millions of tumor-associated plasma DNA end coordinates in the genome. Abundance of plasma DNA molecules with tumor-associated DNA ends correlated with the tumor DNA fractions even in plasma samples of hepatocellular carcinoma patients that were subjected to shallow-depth sequencing analysis. Plasma DNA end coordinates may therefore serve as hallmarks of ctDNA that could be sampled readily and, hence, may improve the cost-effectiveness of liquid biopsy assessment.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
The current diagnosis and monitoring of bladder cancer are heavily reliant on cystoscopy, an invasive and costly procedure. Previous efforts in urine-based detection of bladder cancer focused on ...targeted approaches that are predicated on the tumor expressing specific aberrations. We aimed to noninvasively detect bladder cancer by the genome-wide assessment of methylomic and copy number aberrations (CNAs). We also investigated the size of tumor cell-free (cf)DNA fragments.
Shallow-depth paired-end genome-wide bisulfite sequencing of urinary cfDNA was done for 46 bladder cancer patients and 39 cancer-free controls with hematuria. We assessed (
) proportional contribution from different tissues by methylation deconvolution, (
) global hypomethylation, (
) CNA, and (
) cfDNA size profile.
Methylomic and copy number approaches were synergistically combined to detect bladder cancer with a sensitivity of 93.5% (84.2% for low-grade nonmuscle-invasive disease) and a specificity of 95.8%. The prevalence of methylomic and CNAs reflected disease stage and tumor size. Sampling over multiple time points could assess residual disease and changes in tumor load. Muscle-invasive bladder cancer was associated with a higher proportion of long cfDNA, as well as longer cfDNA fragments originating from genomic regions enriched for tumor DNA.
Bladder cancer can be detected noninvasively in urinary cfDNA by methylomic and copy number analysis without previous knowledge or assumptions of specific aberrations. Such analysis could be used as a liquid biopsy to aid diagnosis and for potential longitudinal monitoring of tumor load. Further understanding of the differential size and fragmentation of cfDNA could improve the detection of bladder cancer.
The addition of increasing concentrations of DNase1 to knockout mice plasma caused increases in the DNase activity in a dose-dependent manner (Mann-Whitney U-test P = 0.07 and 0.03 after addition of2 ...U and 5 U of DNase1, respectively). Overhangs would be obscured by the end-repair step, and short fragments arising from nicks might be lost entirely during the dsDNA library preparation process. ...we also employed a template-switching single-stranded (ss) DNA library preparation protocol (DNA SMART ChIP-Seq Kit, Clontech) to sequence plasma from 2 wildtype and 2 knockout mice to a mean of 6.4X coverage of the haploid genome. ...generation noninvasive fetal genome analysis revealsde novo mutations, single-base parental inheritance, and preferred DNA ends.
Objectives/Hypothesis
Ultrasonography is an emerging clinical tool to study the dysfunction of swallowing muscles. This was the first sonographic study to assess the relationship between suprahyoid ...muscle contraction, hyoid bone displacement, and penetration‐aspiration status (PAS) during swallowing in nasopharyngeal carcinoma (NPC) patients treated with radiotherapy (RT). The study also aimed to establish reliability data for the sonographic technique described.
Study Design
Cross‐sectional study.
Methods
Geniohyoid muscle contraction was quantified using brightness‐mode ultrasonography in this study of 40 post‐RT NPC patients. A series of physiological parameters and PAS were measured using videofluoroscopy.
Results
Intra‐ and inter‐rater agreement values ranged from 0.75 to 0.96 across various sonographic measurements. Percentage increase in the cross‐sectional area of the geniohyoid muscle correlated with anterior (r = 0.42, P < .05) but not superior (r = 0.27, P = .09) hyoid displacement. Anterior hyoid displacement and pharyngeal constriction ratio were significantly associated with PAS score.
Conclusions
Sonographic measurement of suprahyoid muscles provides valuable information on muscle function and is potentially a useful clinical tool in swallowing assessment. Further research is needed to refine the role of this examination in dysphagia.
Level of Evidence
2b. Laryngoscope, 2552–2559, 2018
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Urinary cell-free (cf) DNA holds great potential as a completely noninvasive form of liquid biopsy. Knowledge of the composition of cfDNA by tissue of origin is useful for guiding its clinical uses. ...We conducted a global survey of urinary cfDNA composition using genomewide bisulfite sequencing. While previous studies focused on detecting cfDNA from a single source at a time, genomewide tissue specific methylation signatures allow us to simultaneously deduce the proportional contribution from each contributing tissue. The proportional contributions derived from methylation deconvolution are highly correlated with those calculated using allograft-derived donor-specific genetic markers in the urine of hematopoetic stem cell and renal transplant recipients. We found a large variation of proportional contributions from different tissues. We then assessed if cfDNA undergoes time-dependent fragmentation in urine by conducting in vitro incubation experiments. In vitro incubation at 37°C showed that urinary cfDNA concentration decreased under first order kinetics with a half-life of 2.6 to 5.1h. This is reflected in parallel by a decrease in the proportion of long fragments and increase in amplitude of 10bp periodicity seen in the cfDNA size profile. This global survey of urinary cfDNA has deepened our understanding of the composition, degradation and variation of cfDNA in the urinary tract and has laid a foundation for the use of genomewide urinary cfDNA sequencing as a molecular diagnostics tool.
•Genomewide methylation signatures can infer the tissue of origin of urinary cfDNA.•Large variation in the proportional contribution of cfDNA from different tissues•Urinary cfDNA is fragmented under first-order kinetics in a time dependent manner.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Purpose To determine the changes in corneal optical performance after posterior lamellar corneal transplantation. Design Retrospective case series. Methods The anterior segment in four eyes of four ...patients who underwent Descemet stripping endothelial keratoplasty (DSEK) with cataract extraction and intraocular lens (IOL) implantation were imaged with the Visante anterior segment optical coherence tomography OCT (Carl Zeiss Meditec, Dublin, California, USA). The curvature of the posterior surface of the donor graft was compared with that of the host cornea, and corneal thickness was measured. Results All eyes had a hyperopic refractive error after surgery. The posterior corneal curvature after surgery was more than that before surgery. Average preoperative keratometry was 43.4 diopters (D), and after surgery, it was 42.8 D using keratometry. However, when the postsurgical corneal power was calculated using the Gaussian optics method, the average value was 40.8 D. Conclusions The addition of a donor corneal graft to the posterior surface of decompensated corneas may lessen the effective optical power of the cornea and may have implications for IOL power calculations in these eyes.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Background
To investigate a novel velopharyngeal squeeze maneuver (VPSM) and novel endoscopic pharyngeal contraction grade (EPCG) scale for the evaluation of pharyngeal motor function.
Methods
During ...endoscopic examination of 77 post‐irradiated nasopharyngeal carcinoma patients and control subjects, VPSM was rated and lateral pharyngeal wall movement graded with EPCG scale during swallowing. Pharyngeal constriction ratio (PCR) measured by videofluoroscopy was used for correlation.
Results
VPSM and EPCG scale showed almost perfect intra‐rater and inter‐rater reliability (Kappa: >0.90). VPSM was present in 61% of patients suggesting good pharyngeal motor function. VPSM was predictive of EPCG scale (Wald statistic = 29.99, p < 0.001). EPCG scale also correlated strongly with PCR (r: 0.812) and was predictive for aspiration (odds ratio: 22.14 95% CI 5.01–97.89, p < 0.001).
Conclusions
VPSM and EPCG scale are two novel tools to assess pharyngeal motor function, and both correlate well with pharyngeal contractility and aspiration.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
The term metaverse is described as the next iteration of the Internet. Metaverse is a virtual platform that uses extended reality technologies, i.e. augmented reality, virtual reality, mixed reality, ...3D graphics, and other emerging technologies to allow real-time interactions and experiences in ways that are not possible in the physical world. Companies have begun to notice the impact of the metaverse and how it may help maximize profits. The purpose of this paper is to offer perspectives on several important areas, i.e. marketing, tourism, manufacturing, operations management, education, the retailing industry, banking services, healthcare, and human resource management that are likely to be impacted by the adoption and use of a metaverse. Each includes an overview, opportunities, challenges, and a potential research agenda.
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BFBNIB, GIS, IJS, KISLJ, NUK, PNG, UL, UM, UPUK
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