Interleukin-10 receptor signaling promotes the maintenance of a PD-1int TCF-1+ CD8+ T cell population that sustains anti-tumor immunity.

T cell exhaustion limits anti-tumor immunity and responses to immunotherapy. Here, we explored the microenvironmental signals regulating T cell exhaustion using a model of chronic lymphocytic leukemia (CLL). Single-cell analyses identified a subset of PD-1hi, functionally impaired CD8+ T cells that accumulated in secondary lymphoid organs during disease progression and a functionally competent PD-1int subset. Frequencies of PD-1int TCF-1+ CD8+ T cells decreased upon Il10rb or Stat3 deletion, leading to accumulation of PD-1hi cells and accelerated tumor progression. Mechanistically, inhibition of IL-10R signaling altered chromatin accessibility and disrupted cooperativity between the transcription factors NFAT and AP-1, promoting a distinct NFAT-associated program. Low IL10 expression or loss of IL-10R-STAT3 signaling correlated with increased frequencies of exhausted CD8+ T cells and poor survival in CLL and in breast cancer patients. Thus, balance between PD-1hi, exhausted CD8+ T cells and functional PD-1int TCF-1+ CD8+ T cells is regulated by cell-intrinsic IL-10R signaling, with implications for immunotherapy.

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.

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.

Accurate identification of structural variations from cancer samples.

Structural variations (SVs) are commonly found in cancer genomes. They can cause gene amplification, deletion and fusion, among other functional consequences. With an average read length of hundreds of kilobases, nano-channel-based optical DNA mapping is powerful in detecting large SVs. However, existing SV calling methods are not tailored for cancer samples, which have special properties such as mixed cell types and sub-clones. Here we propose the Cancer Optical Mapping for detecting Structural Variations (COMSV) method that is specifically designed for cancer samples. It shows high sensitivity and specificity in benchmark comparisons. Applying to cancer cell lines and patient samples, COMSV identifies hundreds of novel SVs per sample.

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.

New USPSTF Breast Cancer Screening Recommendations: A Talk With Task Force Vice Chair Dr John Wong.

In this video article, Dr. John B. Wong, Vice Chair of the U.S. Preventive Services Task Force (USPSTF), discusses implications of the new USPSTF recommendation for biennial screening mammography for women 40-74 years old.

In this video article, Dr. John B. Wong, Vice Chair of the U.S. Preventive Services Task Force (USPSTF), discusses implications of the new USPSTF recommendation for biennial screening mammography for women 40­74 years old.

Comparison of clinical outcome between laparoscopic and open hepatectomy of high difficulty score for hepatocellular carcinoma: a propensity score analysis.

BACKGROUND: Laparoscopic liver resection (LLR) of high difficulty score is technically challenging. There is a lack of clinical evidence to support its applicability in terms of the long-term survival benefits. This study aims to compare clinical outcomes between LLR and the open liver resection of high difficulty score for hepatocellular carcinoma (HCC). MATERIALS AND METHODS: From 2010 to 2020, using Iwate criteria, 424 patients underwent liver resection of high difficulty score by the laparoscopic (n = 65) or open (n = 359) approach. Propensity score (PS) matching was performed between the two groups. Short-term and long-term outcomes were compared between PS-matched groups. Univariate and multivariate analyses were performed to identify prognostic factors affecting survival. RESULTS: The laparoscopic group had significantly fewer severe complications (3% vs. 10.8%), and shorter median hospital stays (6 days vs. 8 days) than the open group. Meanwhile, the long-term oncological outcomes were comparable between the two groups, in terms of the tumor recurrence rate (40% vs. 46.1%), the 5-year overall survival rate (75.4% vs. 76.2%), and the 5-year recurrence-free survival rate (50.3% vs. 53.5%). The high preoperative serum alpha-fetoprotein level, multiple tumors, and severe postoperative complications were the independent poor prognostic factors associated with worse overall survival. The surgical approach (Laparoscopic vs. Open) did not influence the survival. CONCLUSION: LLR of high difficulty score for selected patients with HCC has better short-term outcomes than the open approach. More importantly, it can achieve similar long-term survival outcomes as the open approach.

Physician Approaches to Antithrombotic Therapies for Recently Symptomatic Carotid Stenosis.

BACKGROUND: Whereas the beneficial effect of antiplatelet therapy for recurrent stroke prevention has been well established, uncertainties remain regarding the optimal antithrombotic regimen for recently symptomatic carotid stenosis. We sought to explore the approaches of stroke physicians to antithrombotic management of patients with symptomatic carotid stenosis. METHODS: We employed a qualitative descriptive methodology to explore the decision-making approaches and opinions of physicians regarding antithrombotic regimens for symptomatic carotid stenosis. We conducted semi-structured interviews with a purposive sample of 22 stroke physicians (11 neurologists, 3 geriatricians, 5 interventional-neuroradiologists, and 3 neurosurgeons) from 16 centers on four continents to discuss symptomatic carotid stenosis management. We then conducted thematic analysis on the transcripts. RESULTS: Important themes revealed from our analysis included limitations of existing clinical trial evidence, competing surgeon versus neurologist/internist preferences, and the choice of antiplatelet therapy while awaiting revascularization. There was a greater concern for adverse events while using multiple antiplatelet agents (e.g., dual-antiplatelet therapy (DAPT)) in patients undergoing carotid endarterectomy compared to carotid artery stenting. Regional variations included more frequent use of single antiplatelet agents among European participants. Areas of uncertainty included antithrombotic management if already on an antiplatelet agent, implications of nonstenotic features of carotid disease, the role of newer antiplatelet agents or anticoagulants, platelet aggregation testing, and timing of DAPT. CONCLUSION: Our qualitative findings can help physicians critically examine the rationale underlying their own antithrombotic approaches to symptomatic carotid stenosis. Future clinical trials may wish to accommodate identified variations in practice patterns and areas of uncertainty to better inform clinical practice.

Genome-wide detection of cytosine methylation by single molecule real-time sequencing.

5-Methylcytosine (5mC) is an important type of epigenetic modification. Bisulfite sequencing (BS-seq) has limitations, such as severe DNA degradation. Using single molecule real-time sequencing, we developed a methodology to directly examine 5mC. This approach holistically examined kinetic signals of a DNA polymerase (including interpulse duration and pulse width) and sequence context for every nucleotide within a measurement window, termed the holistic kinetic (HK) model. The measurement window of each analyzed double-stranded DNA molecule comprised 21 nucleotides with a cytosine in a CpG site in the center. We used amplified DNA (unmethylated) and M.SssI-treated DNA (methylated) (M.SssI being a CpG methyltransferase) to train a convolutional neural network. The area under the curve for differentiating methylation states using such samples was up to 0.97. The sensitivity and specificity for genome-wide 5mC detection at single-base resolution reached 90% and 94%, respectively. The HK model was then tested on human-mouse hybrid fragments in which each member of the hybrid had a different methylation status. The model was also tested on human genomic DNA molecules extracted from various biological samples, such as buffy coat, placental, and tumoral tissues. The overall methylation levels deduced by the HK model were well correlated with those by BS-seq (r = 0.99; P < 0.0001) and allowed the measurement of allele-specific methylation patterns in imprinted genes. Taken together, this methodology has provided a system for simultaneous genome-wide genetic and epigenetic analyses.

Effectiveness of an Intervention to Improve Decision Making for Older Patients With Advanced Chronic Kidney Disease : A Randomized Controlled Trial.

BACKGROUND: Older patients with advanced chronic kidney disease (CKD) face difficult decisions about managing kidney failure, frequently experiencing decisional conflict, regret, and treatment misaligned with preferences. OBJECTIVE: To assess whether a decision aid about kidney replacement therapy improved decisional quality compared with usual care. DESIGN: Multicenter, randomized, controlled trial. (ClinicalTrials.gov: NCT03522740). SETTING: 8 outpatient nephrology clinics associated with 4 U.S. centers. PARTICIPANTS: English-fluent patients, 70 years and older with nondialysis CKD stages 4 to 5 recruited from 2018 to 2020. INTERVENTION: DART (Decision-Aid for Renal Therapy) is an interactive, web-based decision aid for older adults with CKD. Both groups received written education about treatments. MEASUREMENTS: Change in the decisional conflict scale (DCS) score from baseline to 3, 6, 12, and 18 months. Secondary outcomes included change in prognostic and treatment knowledge and change in uncertainty. RESULTS: Among 400 participants, 363 were randomly assigned: 180 to usual care, 183 to DART. Decisional quality improved with DART with mean DCS declining compared with control (mean difference, -8.5 [95% CI, -12.0 to -5.0]; P < 0.001), with similar findings at 6 months, attenuating thereafter. At 3 months, knowledge improved with DART versus usual care (mean difference, 7.2 [CI, 3.7 to 10.7]; P < 0.001); similar findings at 6 months were modestly attenuated at 18 months (mean difference, 5.9 [CI, 1.4 to 10.3]; P = 0.010). Treatment preferences changed from 58% "unsure" at baseline to 28%, 20%, 23%, and 14% at 3, 6, 12, and 18 months, respectively, with DART, versus 51% to 38%, 35%, 32%, and 18% with usual care. LIMITATION: Latinx patients were underrepresented. CONCLUSION: DART improved decision quality and clarified treatment preferences among older adults with advanced CKD for 6 months after the DART intervention. PRIMARY FUNDING SOURCE: Patient-Centered Outcomes Research Institute (PCORI).

Evaluation of activities of daily living using an electronic version of the Longshi Scale in patients with stroke: reliability, consistency, and preference.

BACKGROUND: The Longshi Scale is a pictorial assessment tool for evaluating activities of daily living (ADL) in patients with stroke. The paper-based version presents challenges; thus, the WeChat version was created to enhance accessibility. Herein, we aimed to validate the inter-rater and test-retest reliabilities of the WeChat version of the Longshi Scale and explore its potential clinical applications. METHODS: We recruited 115 patients with stroke in the study. The ADL results of each patient were assessed using both the WeChat and paper-based version of the Longshi Scale; each evaluation was conducted by 28 health professionals and 115 caregivers separately. To explore the test-retest reliability of the WeChat version, 22 patients were randomly selected and re-evaluated by health professionals using the WeChat version. All evaluation criteria were recorded, and all evaluators were surveyed to indicate their preference between the two versions. RESULTS: Consistency between WeChat and the paper-based Longshi Scale was high for ADL scores by health professionals (ICC2,1 = 0.803-0.988) and caregivers (ICC2,1 = 0.845-0.983), as well as for degrees of disability (κw = 0.870 by professionals; κw = 0.800 by caregivers). Bland-Altman analysis showed no significant discrepancies. The WeChat version exhibited good test-retest reliability (κw = 0.880). The WeChat version showed similar inter-rater reliability in terms of the ADL score evaluated using the paper-based version (ICC2,1 = 0.781-0.941). The time to complete assessments did not differ significantly, although the WeChat version had a shorter information entry time (P < 0.001, 95% confidence interval: -43.463 to -15.488). Health professionals favored the WeChat version (53.6%), whereas caregivers had no significant preference. CONCLUSIONS: The WeChat version of the Longshi Scale is reliable and serves as a suitable alternative for health professionals and caregivers to assess ADL levels in patients with stroke. The WeChat version of the Longshi Scale is considered user-friendly by health professionals, although it is not preferred by caregivers. TRIAL REGISTRATION: This study was approved by the Ethics Committee of the Second People's Hospital of Shenzhen (approval number: 20210812003-FS01) and registered on the Clinical Trial Register Center website: clinicaltrials.gov on January 31, 2022 (registration no.: NCT05214638).

Effectiveness of educational interventions in reducing the stigma of healthcare professionals and healthcare students towards mental illness: A systematic review and meta-analysis.

AIM: To examine the effectiveness of educational interventions in reducing stigma among healthcare professionals and students towards people with mental illness. DESIGN: A systematic review and meta-analysis of randomized controlled trials (RCTs) and cluster RCTs. DATA SOURCES: Articles published from database inception to October 2023 were systematically searched from seven databases (CINAHL, Embase, ProQuest Dissertations and Theses Global, PsycINFO, PubMed, Scopus, Web of Science), following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. METHODS: Random-effect meta-analyses were conducted. Heterogeneity was evaluated using the I2 statistics and Cochran's Q chi-squared test. A quality appraisal conducted at the study level used the Cochrane risk of bias tool and an outcome-level quality assessment utilized the Grades of Recommendation, Assessment, Development and Evaluation Approach. Publication bias was assessed using the funnel plot. RESULTS: Twenty-five articles were included in this review. Meta-analysis reported statistically significant medium and small effect sizes for attitudes towards mental illness and attitudes towards people with mental illness respectively, showing the association between educational interventions and improved attitudes among healthcare professionals and students. However, a statistically non-significant effect was reported for knowledge of mental illness. Subgroup analyses indicated that face-to-face and contact-based interventions were particularly effective at reducing stigma. Notably, single-session interventions were just as effective as multiple sessions, suggesting a potential for resource-efficient approaches. CONCLUSION: Educational interventions demonstrate promise in fostering more positive attitudes towards mental health issues. Future research should aim to determine the long-term effects of these interventions and include patient feedback on the stigmatizing behaviours of healthcare professionals and students, to holistically evaluate the effect of interventions. NO PATIENT OR PUBLIC CONTRIBUTION: This study is a secondary review and does not require relevant contributions from patients or the public. WHAT DOES THIS PAPER CONTRIBUTE TO THE WIDER GLOBAL CLINICAL COMMUNITY?: Face-to-face contact-based educational sessions have proven to be the most effective. Reinforcing learning may be achieved through a series of repeated single-session interventions.

Screening for Speech and Language Delay and Disorders in Children: US Preventive Services Task Force Recommendation Statement.

Importance: Speech and language delays and disorders can pose significant problems for children and their families. Evidence suggests that school-aged children with speech or language delays may be at increased risk of learning and literacy disabilities, including difficulties with reading and writing. Objective: The US Preventive Services Task Force (USPSTF) commissioned a systematic review to evaluate benefits and harms of screening for speech and language delay and disorders in children 5 years or younger. Population: Asymptomatic children 5 years or younger whose parents or clinicians do not have specific concerns about their speech, language, hearing, or development. Evidence Assessment: The USPSTF concludes that the current evidence is insufficient to assess the balance of benefits and harms of screening for speech and language delay and disorders in children who do not present with signs or symptoms or parent/caregiver concerns. Recommendation: The USPSTF concludes that the current evidence is insufficient to assess the balance of benefits and harms of screening for speech and language delay and disorders in children 5 years or younger without signs or symptoms. (I statement).

Interventions to Prevent Falls in Community-Dwelling Older Adults: US Preventive Services Task Force Recommendation Statement.

Importance: Falls are the leading cause of injury-related morbidity and mortality among older adults in the US. In 2018, 27.5% of community-dwelling adults 65 years or older reported at least 1 fall in the past year and 10.2% reported a fall-related injury. In 2021, an estimated 38 742 deaths resulted from fall-related injuries. Objective: The US Preventive Services Task Force (USPSTF) commissioned a systematic review to evaluate the effectiveness and harms of primary care-relevant interventions to prevent falls and fall-related morbidity and mortality in community-dwelling adults 65 years or older. Population: Community-dwelling adults 65 years or older at increased risk of falls. Evidence Assessment: The USPSTF concludes with moderate certainty that exercise interventions provide a moderate net benefit in preventing falls and fall-related morbidity in older adults at increased risk for falls. The USPSTF concludes with moderate certainty that multifactorial interventions provide a small net benefit in preventing falls and fall-related morbidity in older adults at increased risk for falls. Recommendation: The USPSTF recommends exercise interventions to prevent falls in community-dwelling adults 65 years or older who are at increased risk for falls. (B recommendation) The USPSTF recommends that clinicians individualize the decision to offer multifactorial interventions to prevent falls to community-dwelling adults 65 years or older who are at increased risk for falls. Existing evidence indicates that the overall net benefit of routinely offering multifactorial interventions to prevent falls is small. When determining whether this service is appropriate for an individual, patients and clinicians should consider the balance of benefits and harms based on the circumstances of prior falls, presence of comorbid medical conditions, and the patient's values and preferences. (C recommendation).

Interventions for High Body Mass Index in Children and Adolescents: US Preventive Services Task Force Recommendation Statement.

Importance: Approximately 19.7% of children and adolescents aged 2 to 19 years in the US have a body mass index (BMI) at or above the 95th percentile for age and sex, based on Centers for Disease Control and Prevention growth charts from 2000. The prevalence of high BMI increases with age and is higher among Hispanic/Latino, Native American/Alaska Native, and non-Hispanic Black children and adolescents and children from lower-income families. Objective: The US Preventive Services Task Force (USPSTF) commissioned a systematic review to evaluate the evidence on interventions (behavioral counseling and pharmacotherapy) for weight loss or weight management in children and adolescents that can be provided in or referred from a primary care setting. Population: Children and adolescents 6 years or older. Evidence Assessment: The USPSTF concludes with moderate certainty that providing or referring children and adolescents 6 years or older with a high BMI to comprehensive, intensive behavioral interventions has a moderate net benefit. Recommendation: The USPSTF recommends that clinicians provide or refer children and adolescents 6 years or older with a high BMI (≥95th percentile for age and sex) to comprehensive, intensive behavioral interventions. (B recommendation).

Screening for Breast Cancer: US Preventive Services Task Force Recommendation Statement.

Importance: Among all US women, breast cancer is the second most common cancer and the second most common cause of cancer death. In 2023, an estimated 43 170 women died of breast cancer. Non-Hispanic White women have the highest incidence of breast cancer and non-Hispanic Black women have the highest mortality rate. Objective: The USPSTF commissioned a systematic review to evaluate the comparative effectiveness of different mammography-based breast cancer screening strategies by age to start and stop screening, screening interval, modality, use of supplemental imaging, or personalization of screening for breast cancer on the incidence of and progression to advanced breast cancer, breast cancer morbidity, and breast cancer-specific or all-cause mortality, and collaborative modeling studies to complement the evidence from the review. Population: Cisgender women and all other persons assigned female at birth aged 40 years or older at average risk of breast cancer. Evidence Assessment: The USPSTF concludes with moderate certainty that biennial screening mammography in women aged 40 to 74 years has a moderate net benefit. The USPSTF concludes that the evidence is insufficient to determine the balance of benefits and harms of screening mammography in women 75 years or older and the balance of benefits and harms of supplemental screening for breast cancer with breast ultrasound or magnetic resonance imaging (MRI), regardless of breast density. Recommendation: The USPSTF recommends biennial screening mammography for women aged 40 to 74 years. (B recommendation) The USPSTF concludes that the current evidence is insufficient to assess the balance of benefits and harms of screening mammography in women 75 years or older. (I statement) The USPSTF concludes that the current evidence is insufficient to assess the balance of benefits and harms of supplemental screening for breast cancer using breast ultrasonography or MRI in women identified to have dense breasts on an otherwise negative screening mammogram. (I statement).

scSNPdemux: a sensitive demultiplexing pipeline using single nucleotide polymorphisms for improved pooled single-cell RNA sequencing analysis.

BACKGROUND: Here we present scSNPdemux, a sample demultiplexing pipeline for single-cell RNA sequencing data using natural genetic variations in humans. The pipeline requires alignment files from Cell Ranger (10× Genomics), a population SNP database and genotyped single nucleotide polymorphisms (SNPs) per sample. The tool works on sparse genotyping data in VCF format for sample identification. RESULTS: The pipeline was tested on both single-cell and single-nuclei based RNA sequencing datasets and showed superior demultiplexing performance over the lipid-based CellPlex and Multi-seq sample multiplexing technique which incurs additional single cell library preparation steps. Specifically, our pipeline demonstrated superior sensitivity and specificity in cell-identity assignment over CellPlex, especially on immune cell types with low RNA content. CONCLUSIONS: We designed a streamlined pipeline for single-cell sample demultiplexing, aiming to overcome common problems in multiplexing samples using single cell libraries which might affect data quality and can be costly.

Detection and characterization of jagged ends of double-stranded DNA in plasma.

Cell-free DNA in plasma has been used for noninvasive prenatal testing and cancer liquid biopsy. The physical properties of cell-free DNA fragments in plasma, such as fragment sizes and ends, have attracted much recent interest, leading to the emerging field of cell-free DNA fragmentomics. However, one aspect of plasma DNA fragmentomics as to whether double-stranded plasma molecules might carry single-stranded ends, termed a jagged end in this study, remains underexplored. We have developed two approaches for investigating the presence of jagged ends in a plasma DNA pool. These approaches utilized DNA end repair to introduce differential methylation signals between the original sequence and the jagged ends, depending on whether unmethylated or methylated cytosines were used in the DNA end-repair procedure. The majority of plasma DNA molecules (87.8%) were found to bear jagged ends. The jaggedness varied according to plasma DNA fragment sizes and appeared to be in association with nucleosomal patterns. In the plasma of pregnant women, the jaggedness of fetal DNA molecules was higher than that of the maternal counterparts. The jaggedness of plasma DNA correlated with the fetal DNA fraction. Similarly, in the plasma of cancer patients, tumor-derived DNA molecules in patients with hepatocellular carcinoma showed an elevated jaggedness compared with nontumoral DNA. In mouse models, knocking out of the Dnase1 gene reduced jaggedness, whereas knocking out of the Dnase1l3 gene enhanced jaggedness. Hence, plasma DNA jagged ends represent an intrinsic property of plasma DNA and provide a link between nuclease activities and the fragmentation of plasma DNA.

Comparison of Single Molecule, Real-Time Sequencing and Nanopore Sequencing for Analysis of the Size, End-Motif, and Tissue-of-Origin of Long Cell-Free DNA in Plasma.

BACKGROUND: Recent studies using single molecule, real-time (SMRT) sequencing revealed a substantial population of analyzable long cell-free DNA (cfDNA) in plasma. Potential clinical utilities of such long cfDNA in pregnancy and cancer have been demonstrated. However, the performance of different long-read sequencing platforms for the analysis of long cfDNA remains unknown. METHODS: Size biases of SMRT sequencing by Pacific Biosciences (PacBio) and nanopore sequencing by Oxford Nanopore Technologies (ONT) were evaluated using artificial mixtures of sonicated human and mouse DNA of different sizes. cfDNA from plasma samples of pregnant women at different trimesters, hepatitis B carriers, and patients with hepatocellular carcinoma were sequenced with the 2 platforms. RESULTS: Both platforms showed biases to sequence longer (1500 bp vs 200 bp) DNA fragments, with PacBio showing a stronger bias (5-fold overrepresentation of long fragments vs 2-fold in ONT). Percentages of cfDNA fragments 500 bp were around 6-fold higher in PacBio compared with ONT. End motif profiles of cfDNA from PacBio and ONT were similar, yet exhibited platform-dependent patterns. Tissue-of-origin analysis based on single-molecule methylation patterns showed comparable performance on both platforms. CONCLUSIONS: SMRT sequencing generated data with higher percentages of long cfDNA compared with nanopore sequencing. Yet, a higher number of long cfDNA fragments eligible for the tissue-of-origin analysis could be obtained from nanopore sequencing due to its much higher throughput. When analyzing the size and end motif of cfDNA, one should be aware of the analytical characteristics and possible biases of the sequencing platforms being used.

Development and Validation of the Rating of CKD Knowledge Among Older Adults (Know-CKD) With Kidney Failure.

RATIONALE & OBJECTIVE: Few older adults with kidney failure engage in shared decision making (SDM) for kidney replacement therapy. The lack of instruments to assess SDM-relevant knowledge domains may contribute to this. We assessed the reliability and validity of a new instrument, the Rating of CKD Knowledge Older Adults (Know-CKD). STUDY DESIGN: Multistage process, including a stakeholder-engaged development phase, pilot testing, and validation of a knowledge instrument using a cross-sectional survey of older adults with CKD. SETTING & PARTICIPANTS: 363 patients aged 70+years with nondialysis advanced chronic kidney disease (CKD) (estimated glomerular filtration rate [eGFR]<30mL/min/1.73m2) in Boston, Chicago, Portland, ME, and San Diego from June 2018 and January 2020. EXPOSURE: Educational level, higher literacy (Single Item Literacy Screener [SILS]) and numeracy (Subjective Numeracy Scale [SNS]), having participated in clinic-sponsored dialysis education, and self-reported "feeling informed" about options for treatment. OUTCOME: Validity and reliability of the Know-CKD instrument. ANALYTICAL APPROACH: Reliability was assessed with the Kuder-Richardson-20 coefficient. Construct validity was demonstrated by testing a priori hypotheses using t test, analysis of variance (ANOVA) tests, and linear regression analyses. RESULTS: The mean (± SD) participant age was 77.6±5.9 years, and mean eGFR was 22.7±7.2mL/min/1.73m2; 281 participants (78%) self-reported as White. The 12-item Know-CKD assessment had good reliability (Kuder-Richardson-20 reliability coefficient=0.75), and a mean score of 58.2% ± 22.3 SD. The subscales did not attain acceptable reliability. The proportion answering correctly on each item ranged from 20.1% to 91.7%. In examining construct validity, the hypothesized associations held; Know-CKD significantly associated with higher education (ß=6.98 [95% CI, 1.34-12.61], P=0.02), health literacy (ß = -12.67 [95% CI, -19.49 to-5.86], P≤0.001), numeracy per 10% higher (ß=1.85 [95% CI, 1.02-2.69], P≤0.001), and attendance at dialysis class (ß=18.28 [95% CI, 13.30-23.27], P≤0.001). These associations were also observed for the subscales except for prognosis (not associated with literacy or numeracy). LIMITATIONS: Know-CKD is only available in English and has been used only in research settings. CONCLUSIONS: For older adults facing dialysis initiation decisions, Know-CKD is a valid, reliable, and easy to administer measure of knowledge. Further research should examine the relationship of kidney disease knowledge and SDM, patient satisfaction, and clinical outcomes. PLAIN-LANGUAGE SUMMARY: The Rating of CKD Knowledge Among Older Adults (Know-CKD) study measures knowledge of chronic kidney disease (CKD) and is designed for older adults. Most existing knowledge measures for CKD focus on people of all ages and all CKD stages. This measure is useful because it will allow researchers to assess how well patient education efforts are working. Patient education is a way to help patients make decisions about their care. We describe how the measure was developed by a team of doctors, researchers, and patients, and how the measure performed among persons with advanced CKD aged 70 years and older. Know-CKD can inform efforts to improve shared decision-making research and practice for older patients with kidney disease.