The molecular diversity of dissolved organic matter in forest streams across central Canadian boreal watersheds.

Small headwater streams can mobilize large amounts of terrestrially derived dissolved organic matter (DOM). While the molecular composition of DOM has important controls on biogeochemical cycles and carbon cycling, how stationary landscape metrics affect DOM composition is poorly understood, particularly in relation to non-stationary effects from hydrological changes across seasons. Here, we apply a combination of Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) and absorbance spectroscopy to characterize stream DOM from 13 diverse watersheds across the central Canadian boreal forests and statistically relate DOM compositional characteristics to landscape topography and hydrological metrics. We found that watershed runoff across different surface physiographies produced DOM with distinctly different chemical compositions related to runoff pH. Specifically, streams in sandy soil watersheds contained more abundant aromatic, nitrogenated and sulfurized fractions of DOM, likely due to a combination of lower soil capacity to absorb DOM than other soil types and high conifer forest coverage that generated acidic litterfall in more sandy watersheds. In contrast, streams with more neutral pH in watersheds with shallow soils had DOM resembling low oxidized phenolic molecules mainly due to increased brush/alder and deciduous vegetation coverage in relatively steeper watersheds. However, as precipitation and flows increased in the fall, the overall water chemistry of streams became more similar as runoff pH increased, the overall chemical diversity of DOM in streams decreased, and stream DOM resembled fresher, lower molecular weight lignin material likely originating from freshly produced leaf litter. Together, our findings show that during hydrologically disconnected periods, pH and landscape characteristics have important controls on the mobilization of aromatic DOM but that many landscape-specific characteristics in the Canadian boreal forest are less influential on DOM processing during wetter conditions where chemically similar, plant-derived DOM signatures are preferentially mobilized. These findings collectively help predict the composition of DOM across diverse watersheds in the Canadian boreal to inform microbial and contaminant biogeochemical processes in downstream ecosystems.

Genomic origin, fragmentomics, and transcriptional properties of long cell-free DNA molecules in human plasma.

Recent studies have revealed an unexplored population of long cell-free DNA (cfDNA) molecules in human plasma using long-read sequencing technologies. However, the biological properties of long cfDNA molecules (>500 bp) remain largely unknown. To this end, we have investigated the origins of long cfDNA molecules from different genomic elements. Analysis of plasma cfDNA using long-read sequencing reveals an uneven distribution of long molecules from across the genome. Long cfDNA molecules show overrepresentation in euchromatic regions of the genome, in sharp contrast to short DNA molecules. We observe a stronger relationship between the abundance of long molecules and mRNA gene expression levels, compared with short molecules (Pearson's r = 0.71 vs. -0.14). Moreover, long and short molecules show distinct fragmentation patterns surrounding CpG sites. Leveraging the cleavage preferences surrounding CpG sites, the combined cleavage ratios of long and short molecules can differentiate patients with hepatocellular carcinoma (HCC) from non-HCC subjects (AUC = 0.87). We also investigated knockout mice in which selected nuclease genes had been inactivated in comparison with wild-type mice. The proportion of long molecules originating from transcription start sites are lower in Dffb-deficient mice but higher in Dnase1l3-deficient mice compared with that of wild-type mice. This work thus provides new insights into the biological properties and potential clinical applications of long cfDNA molecules.

Early detection of nasopharyngeal carcinoma: performance of a short contrast-free screening magnetic resonance imaging.

BACKGROUND: Although contrast-enhanced magnetic resonance imaging (MRI) detects early-stage nasopharyngeal carcinoma (NPC) not detected by endoscopic-guided biopsy (EGB), a short contrast-free screening MRI would be desirable for NPC screening programs. This study evaluated a screening MRI in a plasma Epstein-Barr virus (EBV)-DNA NPC screening program. METHODS: EBV-DNA-screen-positive patients underwent endoscopy, and endoscopy-positive patients underwent EGB. EGB was negative if the biopsy was negative or was not performed. Patients also underwent a screening MRI. Diagnostic performance was based on histologic confirmation of NPC in the initial study or during a follow-up period of at least 2 years. RESULTS: The study prospectively recruited 354 patients for MRI and endoscopy; 40/354 (11.3%) endoscopy-positive patients underwent EGB. Eighteen had NPC (5.1%), and 336 without NPC (94.9%) were followed up for a median of 44.8 months. MRI detected additional NPCs in 3/18 (16.7%) endoscopy-negative and 2/18 (11.1%) EGB-negative patients (stage I/II, n = 4; stage III, n = 1). None of the 24 EGB-negative patients who were MRI-negative had NPC. MRI missed NPC in 2/18 (11.1%), one of which was also endoscopy-negative. The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of MRI, endoscopy, and EGB were 88.9%, 91.1%, 34.8%, 99.4%, and 91.0%; 77.8%, 92.3%, 35.0%, 98.7%, and 91.5%; and 66.7%, 92.3%, 31.6%, 98.1%, and 91.0%, respectively. CONCLUSION: A quick contrast-free screening MRI complements endoscopy in NPC screening programs. In EBV-screen-positive patients, MRI enables early detection of NPC that is endoscopically occult or negative on EGB and increases confidence that NPC has not been missed.

Mercury concentrations and export from small central Canadian boreal forest catchments before, during, and after forest harvest.

Northern boreal forests are a strong sink for mercury (Hg), a global contaminant of significant concern to wildlife and human health. Mercury stored in forest soils can be mobilized via runoff and erosion, and under suitable conditions can be methylated to its much more bioaccumulative form, methylmercury. Forest harvesting can affect the mobilization and methylation of Hg, though the direction and magnitude of the impact is unclear or conflicting across previous studies. This study examined 5 harvested and 2 reference watersheds in northwestern Ontario, Canada, before, during, and after harvest to quantify changes in stream total and methylmercury concentration and loads and identified potential landscape and management factors that contribute to differences in stream response. In watersheds where streams were buffered by natural vegetation (≥30 m), no significant changes in total Hg or methylmercury concentrations or loads were observed. Significant increases in methylmercury concentrations and loads were observed downstream of a stream crossing in a watershed where the relatively small stream was unmapped and therefore only buffered by a 3 m machine exclusion zone. These results show that when current best management practices that minimize soil and water disturbance are followed, harvest can have a minimal impact on total and methylmercury loads, even in extensively harvested watersheds. However, there is a need for improved mapping of small streams to ensure best management practices are applied adequately across the landscape.

The application of epiphenotyping approaches to DNA methylation array studies of the human placenta.

BACKGROUND: Genome-wide DNA methylation (DNAme) profiling of the placenta with Illumina Infinium Methylation bead arrays is often used to explore the connections between in utero exposures, placental pathology, and fetal development. However, many technical and biological factors can lead to signals of DNAme variation between samples and between cohorts, and understanding and accounting for these factors is essential to ensure meaningful and replicable data analysis. Recently, "epiphenotyping" approaches have been developed whereby DNAme data can be used to impute information about phenotypic variables such as gestational age, sex, cell composition, and ancestry. These epiphenotypes offer avenues to compare phenotypic data across cohorts, and to understand how phenotypic variables relate to DNAme variability. However, the relationships between placental epiphenotyping variables and other technical and biological variables, and their application to downstream epigenome analyses, have not been well studied. RESULTS: Using DNAme data from 204 placentas across three cohorts, we applied the PlaNET R package to estimate epiphenotypes gestational age, ancestry, and cell composition in these samples. PlaNET ancestry estimates were highly correlated with independent polymorphic ancestry-informative markers, and epigenetic gestational age, on average, was estimated within 4 days of reported gestational age, underscoring the accuracy of these tools. Cell composition estimates varied both within and between cohorts, as well as over very long placental processing times. Interestingly, the ratio of cytotrophoblast to syncytiotrophoblast proportion decreased with increasing gestational age, and differed slightly by both maternal ethnicity (lower in white vs. non-white) and genetic ancestry (lower in higher probability European ancestry). The cohort of origin and cytotrophoblast proportion were the largest drivers of DNAme variation in this dataset, based on their associations with the first principal component. CONCLUSIONS: This work confirms that cohort, array (technical) batch, cell type proportion, self-reported ethnicity, genetic ancestry, and biological sex are important variables to consider in any analyses of Illumina DNAme data. We further demonstrate the specific utility of epiphenotyping tools developed for use with placental DNAme data, and show that these variables (i) provide an independent check of clinically obtained data and (ii) provide a robust approach to compare variables across different datasets. Finally, we present a general framework for the processing and analysis of placental DNAme data, integrating the epiphenotype variables discussed here.

Performance of neuroretinal rim thickness measurement by Cirrus high-definition optical coherence tomography in myopic eyes.

Neuroretinal rim (NRR) measurement can aid the diagnosis of glaucoma. A few studies reported that Cirrus optical coherence tomography (OCT) had NRR segmentation errors. The current study investigated segmentation success of NRR in myopic eyes using the Cirrus built-in software and to determine the number of acquisitions required to identify NRR thinning. Right eye of 87 healthy adult myopes had an optic disc scanned using Cirrus HD-OCT for five successive acquisitions. A masked examiner evaluated 36 radial line images of each scan to screen for segmentation errors using the built-in software at the Bruch's membrane opening (BMO) and/or internal limiting membrane (ILM). Participants with three accurate NRR acquisitions had their average NRR thickness determined. This result was compared with average of the two acquisitions and the first acquisition. Among 435 OCT scans of the optic disc (87 eyes × 5 acquisitions), 129 (29.7%) scans had segmentation errors that occurred mainly at the ILM. The inferior-temporal and superior meridians had slightly more segmentation errors than other meridians, independent of axial length, amount of myopia, or presence of peripapillary atrophy. Sixty-five eyes (74.7%) had at least three accurate NRR measurements. The three acquisitions had high reliability in NRR thickness in the four quadrants (intraclass correlation coefficient > 0.990, coefficient of variation < 3.9%). NRR difference between the first acquisition and the average of three acquisitions was small (mean difference 2 ± 13 µm, 95% limits of agreement within ± 30 µm) among the four quadrants. Segmentation errors in NRR measurements appeared regardless of axial length, amount of myopia, or presence of peripapillary atrophy. Cirrus segmentation lines should be manually inspected when measuring NRR thickness.

Performance and Operational Feasibility of Epstein-Barr Virus-Based Screening for Detection of Nasopharyngeal Carcinoma: Direct Comparison of Two Alternative Approaches.

PURPOSE: Two Epstein-Barr virus (EBV)-based testing approaches have shown promise for early detection of nasopharyngeal carcinoma (NPC). Neither has been independently validated nor their performance compared. We compared their diagnostic performance in an independent population. METHODS: We tested blood samples from 819 incident Taiwanese NPC cases (213 early-stage, American Joint Committee on Cancer version 7 stages I and II) diagnosed from 2010 to 2014 and from 1,768 controls from the same region, frequency matched to cases on age and sex. We compared an EBV antibody score using immunoglobulin A antibodies measured by enzyme-linked immunosorbent assay (EBV antibody score) and plasma EBV DNA load measured by real-time PCR followed by next-generation sequencing (NGS) among EBV DNA-positive individuals (EBV DNA algorithm). RESULTS: EBV antibodies and DNA load were measured for 2,522 (802 cases; 1,720 controls) and 2,542 (797 cases; 1,745 controls) individuals, respectively. Of the 898 individuals positive for plasma EBV DNA and therefore eligible for NGS, we selected 442 (49%) for NGS testing. The EBV antibody score had a sensitivity of 88.4% (95% CI, 86.1 to 90.6) and a specificity of 94.9% (95% CI, 93.8 to 96.0) for NPC. The EBV DNA algorithm yielded significantly higher sensitivity (93.2%; 95% CI, 91.3 to 94.9; P = 1.33 × 10-4) and specificity (98.1%; 95% CI, 97.3 to 98.8; P = 3.53 × 10-7). For early-stage NPC, the sensitivities were 87.1% (95% CI, 82.7 to 92.4) for the EBV antibody score and 87.0% (95% CI, 81.9 to 91.5) for the EBV DNA algorithm (P = .514). For regions with a NPC incidence of 20-100/100,000 person-years (eg, residents in southern China and Hong Kong), these two approaches yielded similar numbers needed to screen (EBV antibody score: 5,656-1,131; EBV DNA algorithm: 5,365-1,073); positive predictive values ranged from 0.4% to 1.7% and 1.0% to 4.7%, respectively. CONCLUSION: We demonstrated high sensitivity and specificity of EBV antibody and plasma EBV DNA for NPC detection, with slightly inferior performance of the EBV antibody score. Cost-effectiveness studies are needed to guide screening implementation.

Fragmentation landscape of cell-free DNA revealed by deconvolutional analysis of end motifs.

Cell-free DNA (cfDNA) fragmentation is nonrandom, at least partially mediated by various DNA nucleases, forming characteristic cfDNA end motifs. However, there is a paucity of tools for deciphering the relative contributions of cfDNA cleavage patterns related to underlying fragmentation factors. In this study, through non-negative matrix factorization algorithm, we used 256 5' 4-mer end motifs to identify distinct types of cfDNA cleavage patterns, referred to as "founder" end-motif profiles (F-profiles). F-profiles were associated with different DNA nucleases based on whether such patterns were disrupted in nuclease-knockout mouse models. Contributions of individual F-profiles in a cfDNA sample could be determined by deconvolutional analysis. We analyzed 93 murine cfDNA samples of different nuclease-deficient mice and identified six types of F-profiles. F-profiles I, II, and III were linked to deoxyribonuclease 1 like 3 (DNASE1L3), deoxyribonuclease 1 (DNASE1), and DNA fragmentation factor subunit beta (DFFB), respectively. We revealed that 42.9% of plasma cfDNA molecules were attributed to DNASE1L3-mediated fragmentation, whereas 43.4% of urinary cfDNA molecules involved DNASE1-mediated fragmentation. We further demonstrated that the relative contributions of F-profiles were useful to inform pathological states, such as autoimmune disorders and cancer. Among the six F-profiles, the use of F-profile I could inform the human patients with systemic lupus erythematosus. F-profile VI could be used to detect individuals with hepatocellular carcinoma, with an area under the receiver operating characteristic curve of 0.97. F-profile VI was more prominent in patients with nasopharyngeal carcinoma undergoing chemoradiotherapy. We proposed that this profile might be related to oxidative stress.

Recommendations for Epstein-Barr virus-based screening for nasopharyngeal cancer in high- and intermediate-risk regions.

A meeting of experts was held in November 2021 to review and discuss available data on performance of Epstein-Barr virus (EBV)-based approaches to screen for early stage nasopharyngeal carcinoma (NPC) and methods for the investigation and management of screen-positive individuals. Serum EBV antibody and plasma EBV DNA testing methods were considered. Both approaches were found to have favorable performance characteristics and to be cost-effective in high-risk populations. In addition to endoscopy, use of magnetic resonance imaging (MRI) to investigate screen-positive individuals was found to increase the sensitivity of NPC detection with minimal impact on cost-effectiveness of the screening program.

Fragment Ends of Circulating Microbial DNA as Signatures for Pathogen Detection in Sepsis.

BACKGROUND: Nuclear-derived cell-free DNA (cfDNA) molecules in blood plasma are nonrandomly fragmented, bearing a wealth of information related to tissues of origin. DNASE1L3 (deoxyribonuclease 1 like 3) is an important player in shaping the fragmentation of nuclear-derived cfDNA molecules, preferentially generating molecules with 5 CC dinucleotide termini (i.e., 5 CC-end motif). However, the fragment end properties of microbial cfDNA and its clinical implication remain to be explored. METHODS: We performed end motif analysis on microbial cfDNA fragments in plasma samples from patients with sepsis. A sequence context-based normalization method was used to minimize the potential biases for end motif analysis. RESULTS: The end motif profiles of microbial cfDNA appeared to resemble that of nuclear cfDNA (Spearman correlation coefficient: 0.82, P value 0.001). The CC-end motif was the most preferred end motif in microbial cfDNA, suggesting that DNASE1L3 might also play a role in the fragmentation of microbe-derived cfDNA in plasma. Of note, differential end motifs were present between microbial cfDNA originating from infection-causing pathogens (enriched at the CC-end) and contaminating microbial DNA potentially derived from reagents or the environment (nearly random). The use of fragment end signatures allowed differentiation between confirmed pathogens and contaminating microbes, with an area under the receiver operating characteristic curve of 0.99. The performance appeared to be superior to conventional analysis based on microbial cfDNA abundance alone. CONCLUSIONS: The use of fragmentomic features could facilitate the differentiation of underlying contaminating microbes from true pathogens in sepsis. This work demonstrates the potential usefulness of microbial cfDNA fragmentomics in metagenomics analysis.

Malignancies in Immunoglobulin G4-related ophthalmic disease.

PURPOSE: Clinical phenotypes in Immunoglobulin G4-related disease (IgG4-RD) according to the patterns of affecting organs have different risks of malignancies. We attempt to determine the association of malignancies with IgG4-related ophthalmic disease (IgG4-ROD). DESIGN: Retrospective cohort study. METHODS: Review of medical records, orbital images and histopathology reports in a territory-wide cohort of biopsy proven IgG4-ROD patients from 2005-2019. FINDINGS: Among 122 patients who had biopsies taken from adnexal lesions including lacrimal glands (n = 108), orbital mass (n = 30), infiltrated orbital fat (n = 10), conjunctiva (n = 2) or extraocular muscles (n = 3), 13% (16/122) developed malignancies over 73 ± 48months' follow-up. There were 9 cases of ocular adnexal lymphoma (OAL) and 7 extra-orbital malignancies. Compared with the general population, the incidence of OAL was significantly higher (standardized incidence ratios, SIRs = 10.0, 95%CI = 4.5-17.6) while that of extra-orbital malignancies was similar. The SIRs was highest within the first year (SIR = 46.7, 95%CI = 18.5-87.6) when 7 OAL were concomitantly diagnosed. Patients who developed OAL or extra-orbital malignancies were older than other patients at IgG4-ROD diagnosis (64.9 ± 7.1, 68.3 ± 8.5 versus 55.2 ± 15.0 years, P < 0.05). Asymmetric lacrimal gland enlargement (78% versus 13%), lack of frontal (0% versus 12%) or infraorbital nerve enlargement (0% versus 36%) were associated with OAL (all P < 0.05). Pre-treatment serum IgG4 level or extra-orbital IgG4-RD involvement was similar among patients with or without malignancies. CONCLUSION: In this biopsy-proven IgG4-ROD cohort, 7% developed OAL which was 10 times higher than the general population. Patients with asymmetric lacrimal gland enlargement or without trigeminal nerves involvement radiologically were associated with OAL.

Plasma Epstein-Barr Virus DNA and Risk of Future Nasopharyngeal Cancer.

EBV DNA Rescreening StudyPatients who had participated in a previous plasma Epstein-Barr virus (EBV) DNA screening study were rescreened. Of the 17,838 rescreened patients, 423 had persistently detectable plasma EBV DNA; 24 of these patients developed nasopharyngeal carcinoma. Sixty-seven percent of them received a diagnosis of early-stage disease and had increased progression-free survival compared with historical controls.

Epigenetic analysis of cell-free DNA by fragmentomic profiling.

Cell-free DNA (cfDNA) fragmentation patterns contain important molecular information linked to tissues of origin. We explored the possibility of using fragmentation patterns to predict cytosine-phosphate-guanine (CpG) methylation of cfDNA, obviating the use of bisulfite treatment and associated risks of DNA degradation. This study investigated the cfDNA cleavage profile surrounding a CpG (i.e., within an 11-nucleotide [nt] window) to analyze cfDNA methylation. The cfDNA cleavage proportion across positions within the window appeared nonrandom and exhibited correlation with methylation status. The mean cleavage proportion was ∼twofold higher at the cytosine of methylated CpGs than unmethylated ones in healthy controls. In contrast, the mean cleavage proportion rapidly decreased at the 1-nt position immediately preceding methylated CpGs. Such differential cleavages resulted in a characteristic change in relative presentations of CGN and NCG motifs at 5' ends, where N represented any nucleotide. CGN/NCG motif ratios were correlated with methylation levels at tissue-specific methylated CpGs (e.g., placenta or liver) (Pearson's absolute r > 0.86). cfDNA cleavage profiles were thus informative for cfDNA methylation and tissue-of-origin analyses. Using CG-containing end motifs, we achieved an area under a receiver operating characteristic curve (AUC) of 0.98 in differentiating patients with and without hepatocellular carcinoma and enhanced the positive predictive value of nasopharyngeal carcinoma screening (from 19.6 to 26.8%). Furthermore, we elucidated the feasibility of using cfDNA cleavage patterns to deduce CpG methylation at single CpG resolution using a deep learning algorithm and achieved an AUC of 0.93. FRAGmentomics-based Methylation Analysis (FRAGMA) presents many possibilities for noninvasive prenatal, cancer, and organ transplantation assessment.

Clinical applications of circulating tumor-derived DNA in the management of gastrointestinal cancers - current evidence and future directions.

Advances in Next Generation Sequencing (NGS) technologies have enabled the accurate detection and quantification of circulating tumor-derived (ct)DNA in most gastrointestinal (GI) cancers. The prognostic and predictive utility of ctDNA in patiets with different stages of colorectal (CRC), gastro-esophageal (GEC) and pancreaticobiliary cancers (PBC) are currently under active investigation. The most mature clinical data to date are derived from studies in the prognostic utility of personalized ctDNA-based NGS assays in the detection of minimal residual disease (MRD) and early recurrence after surgery in CRC and other GI cancers. These findings are being validated in several prospective studies which are designed to test if ctDNA could outperform conventional approaches in guiding adjuvant chemotherapy, and in post-operative surveillance in some GI cancers. Several adaptive studies using ctDNA as a screening platform are also being used to identify patients with actionable genomic alterations for clinical trials of targeted therapies. In the palliative setting, ctDNA monitoring during treatment has shown promise in the detection and tracking of clonal variants associated with acquired resistance to targeted therapies and immune-checkpoint inhibitors (ICI). Moreover, ctDNA may help to guide the therapeutic re-challenge of targeted therapies in patients who have prior exposure to such treatment. This review will examine the most updated research findings on ctDNA as a biomarker in CRC, GEC and PBCs. It aims to provide insights into how the unique strengths of this biomarker could be optimally leveraged in improving the management of these GI cancers.

[Procedural skills training in a developing country: 10 tips]. / Vaardigheidstraining in ontwikkelingslanden: 10 tips.

Procedural skills training in a developing country is challenging, but can improve the quality of care for patients with an important regional health problem. A structured, brief, teach-the-teacher-based training course supports physicians in developing countries to acquire skills needed to perform a complex procedure independently. A case of a patient diagnosed with esophageal cancer is presented and 10 tips are given for training procedural skills in a developing country.