Higher Risk of Tumor Recurrence in NASH-Related Hepatocellular Carcinoma Following Curative Resection

Background: The outcomes for patients with NASH-related HCC after curative resection have not been clarified. This study compared the overall survival (OS), time-to-tumor recurrence (TTR), and recurrence-free survival (RFS) associated with NASH-related HCC and virus-related HCC after resection. Methods: Patients with HCC who underwent curative resection were retrospectively enrolled. Baseline characteristics, including disease etiologies and clinical and tumor features, were reviewed. The primary outcomes were OS, TTR, and RFS. Results: Two hundred and six patients were enrolled (HBV: n = 121, HCV: n = 54, NASH: n = 31). Of those with virus-related HCC, 84.0% achieved viral suppression. In both the overall and propensity-score-matched cohorts, those with NASH-related HCC experienced recurrence significantly earlier than those with virus-related HCC (median TTR: 1108 days vs. non-reached; p = 0.03). Through multivariate analysis, NASH-related HCC (hazard ratio (HR), 2.27; 95% confidence interval (CI), 1.25–4.12) was independently associated with early recurrence. The unadjusted RFS rate of the NASH-related HCC group was lower than the virus-related HCC group. There was no difference in the OS between the two groups. Conclusions: NASH-related HCC was associated with earlier tumor recurrence following curative resection compared to virus-related HCC. Post-surgical surveillance is crucial for detecting early recurrence in patients with NASH-related HCC.     

Rural-urban Disparities in the Prevalence of Mild Cognitive Impairment and Dementia in Taiwan: A Door-to-door Nationwide Study

BackgroundScreening or diagnosis for the elderly with dementia in rural regions might be delayed and underestimated due to limited utilization of healthcare resources. This study aimed to evaluate the disparities of prevalence and risk factors of mild cognitive impairment (MCI) and dementia between urban and rural residence.MethodsIn this nationwide door-to-door survey, 10,432 participants aged 65 years and more were selected through computerized random sampling from all administrative districts in Taiwan and were assessed using an in-person interview. We calculated the prevalence of MCI and dementia, with their risk factors examined using multivariable logistic regression.ResultsThe prevalence of dementia in rural, suburban, and urban areas among the elderly was 8.69% (95% CI, 8.68–8.69), 6.63% (95% CI, 6.62–6.63), and 4.46% (95% CI, 4.46–4.47), respectively. A similar rural-suburban-urban gradient relationship on the dementia prevalence was observed in any age and sex group. The rural:urban ratio was higher in women than in men for both MCI and dementia. Urbanization remained to be an independent factor for both MCI and dementia after adjustment for age, gender, education, lifestyle, and health status. The beneficial effects of exercise on dementia were more evident in rural areas than in urban ones.ConclusionSignificantly higher prevalence of MCI and dementia were found in rural areas than in urban ones, especially for women. The odds of risk factors for MCI and dementia varied by urbanization status. Focus on the rural-urban inequality and the modification of associated factors specifically for different urbanization levels are needed.Key words: dementia, mild cognitive impairment, prevalence, risk factors, urbanization     

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.

Fragmentation patterns of cell-free DNA (cfDNA) molecules contain a wealth of molecular information related to their tissues of origin (1). For instance, compared with the background DNA molecules that are mainly derived from the hematopoietic system (2, 3), size shortening of fetal and tumoral DNA molecules occurs in the plasma DNA of pregnant women and cancer patients, respectively (4–6). In addition, a series of 10-bp periodicities were present in fetal and tumoral DNA molecules below 146 bp, with a relative reduction in the major peak at 166 bp (1). Such characteristic size profiles suggest that the fragmentation of cfDNA may be associated with nucleosome structures (5, 7). Many important characteristics pertaining to cfDNA fragmentation have been unveiled recently, such as nucleosome footprints (8, 9), fragment end motifs (10), preferred ends (7, 11), and jagged ends (12), which are examples of fragmentomic markers (1).cfDNA fragmentomics is an emergent and actively pursued area, with wide-ranging biological and clinical implications. It has been reported that the use of fragmentation patterns of cfDNA could inform the expression status of genes (13, 14). Using mouse models, DNA nucleases (e.g., DNASE1L3) were found to play important roles in the generation of plasma DNA molecules (15, 16). Fragmentomic features, such as cfDNA end motifs and jagged ends, were further demonstrated to be useful for monitoring DNA nuclease activities, providing biomarkers for autoimmune diseases (e.g., systemic lupus erythematosus) (17, 18). In addition, the deficiencies of nuclease activities in a mouse model resulted in altered DNA methylation profiles of plasma DNA molecules (19). However, how cfDNA fragmentation patterns interplay with DNA methylation in human individuals under different pathophysiological conditions, such as pregnancy and oncogenesis, and in healthy patients without nuclease deficiency, is unknown. It is also not known whether fragmentomic features can be used to deduce cfDNA methylation status.A widely employed way to assess DNA methylation is through bisulfite sequencing (20). A key limitation of this approach is the severe degradation of DNA molecules caused by the bisulfite treatment (21), which greatly increases the sampling variation when analyzing rare target molecules (e.g., tumoral cfDNA at early stages of cancer). Many efforts have been made toward overcoming this issue. For example, Vaisvila et al. developed enzymatic methyl sequencing for which DNA molecules were treated using tet methylcytosine dioxygenase 2 and T4 phage β-glucosyltransferase, followed by the apolipoprotein B mRNA editing enzyme catalytic subunit 3A (APOBEC3A) treatment. Cytosine conversion based on enzymatic processes was reported to be much less destructive (22). Recently, researchers developed approaches making use of third-generation sequencing technologies such as single-molecule real-time sequencing (Pacific Biosciences) (23) and nanopore sequencing (24) to analyze cytosine-phosphate-guanine (CpG) methylation patterns in native DNA molecules, theoretically overcoming the above-mentioned limitation. However, compared with second-generation sequencing (also called next-generation sequencing [NGS]) technologies, the throughput of third-generation sequencing technologies is generally lower and the sequencing cost per nucleotide (nt) is much higher, thus restricting its immediate application in clinical settings. Here, we explore the feasibility of enabling the assessment of DNA methylation using fragmentomic characteristics of cfDNA molecules deduced from NGS results without the use of bisulfite or enzymatic treatment. If successful, such an approach could leverage the high throughput of NGS while obviating the use of chemical/enzymatic conversion and could potentially be readily integrated into currently used NGS-based platforms for cfDNA analysis.In this study, we utilize the fragmentation patterns proximal to a CpG site for deducing its methylation status. The fragmentation pattern is depicted by the frequency of cfDNA fragment ends at each position within a certain nt range relative to a CpG of interest, termed a cleavage profile (Fig. 1). Such a cleavage profile varies according to the methylation status of the CpG site of interest, providing the basis for methylation analysis by using fragmentomic features. We further correlated two types of end motifs (CGN and NCG; N represents any nucleotide of A, C, G, or T) resulting from differential cutting in the measurement window related to DNA methylation, attempting to construct a simplified approach for methylation analysis. Modeling CpG methylation using cfDNA fragmentation may facilitate noninvasive prenatal testing, cancer detection, and tissue-of-origin analysis (Fig. 1). Furthermore, we explore the feasibility of using deep learning to deduce the methylation status at single CpG resolution through the cleavage profile (Fig. 1). We refer to this FRAGmentomics-based Methylation Analysis as FRAGMA in this study.Open in a separate windowFig. 1.Schematic for FRAGMA of cfDNA molecules. cfDNA molecules were sequenced by massively parallel sequencing and aligned to the human reference genome. The cleavage proportion within an 11-nt window (the cleavage measurement window) was used to measure the cutting preference of cfDNA molecules. The patterns of cleavage proportion within a window (the cleavage profile) depended on the methylation status of one or more CpG sites associated with that window. For example, a methylated CpG site might confer a higher probability of cfDNA cutting at the cytosine in the CpG context, but an unmethylated site might not. Such methylation-dependent differential fragmentation within a cleavage measurement window resulted in the change in CGN/NCG motif ratio. Thus, the CGN/NCG motif ratio provided a simplified version for reflecting CpG methylation, allowing cfDNA tissue-of-origin analysis of cfDNA and cancer detection. Furthermore, the great number of cleavage profiles derived from cfDNA molecules might provide an opportunity to train a deep learning model for methylation prediction at the single CpG resolution.     

Patterns of biopsy-proven renal diseases in geriatric patients: A single medical center experience

The elderly population is expanding rapidly, and that has become a major healthcare burden in terms of chronic kidney disease. The distribution patterns of kidney diseases in these elderly patients remain largely unclear. Here, we compared biopsy-based renal disease patterns between elderly and nonelderly patients. We performed a single-center, retrospective study (1992–2008) on biopsy-proven renal diseases to compare results between geriatric patients (age ≥ 65 years; n = 254) and nongeriatric patients (18 ≤ age < 65 years; n = 2592). Renal pathology was interpreted by pathologists based on light microscopy, immunofluorescence, and electron microscopy. The ages of the geriatric and nongeriatric groups were 71.8 ± 4.5 (65.1–87.3) and 39.7 ± 17.6 (18–64.9) years, respectively, and 74% and 41% of them, respectively, were men. In the geriatric group, the most frequent diagnosis was membranous nephropathy (46.1%), followed by minimal change disease/focal segmental glomerulosclerosis (16.9%), diabetic nephropathy (8.3%), hypertensive nephrosclerosis (7.5%), and IgA nephropathy (5.9%). The geriatric group had more membranous nephropathy and less lupus nephritis and IgA nephropathy than the nongeriatric group. Furthermore, the 5-year survival rate of the geriatric group was significantly low. Our results demonstrated the different distributions of renal biopsy patterns in geriatric patients diagnosed with acute or chronic progressive kidney injury and proteinuria through renal biopsy.     

Topological crossing in the misfolded Tetrahymena ribozyme resolved by cryo-EM

The Tetrahymena group I intron has been a key system in the understanding of RNA folding and misfolding. The molecule folds into a long-lived misfolded intermediate (M) in vitro, which has been known to form extensive native-like secondary and tertiary structures but is separated by an unknown kinetic barrier from the native state (N). Here, we used cryogenic electron microscopy (cryo-EM) to resolve misfolded structures of the Tetrahymena L-21 ScaI ribozyme. Maps of three M substates (M1, M2, M3) and one N state were achieved from a single specimen with overall resolutions of 3.5 Å, 3.8 Å, 4.0 Å, and 3.0 Å, respectively. Comparisons of the structures reveal that all the M substates are highly similar to N, except for rotation of a core helix P7 that harbors the ribozyme’s guanosine binding site and the crossing of the strands J7/3 and J8/7 that connect P7 to the other elements in the ribozyme core. This topological difference between the M substates and N state explains the failure of 5′-splice site substrate docking in M, supports a topological isomer model for the slow refolding of M to N due to a trapped strand crossing, and suggests pathways for M-to-N refolding.

The Tetrahymena thermophila group I self-splicing intron was the first RNA-only system to be identified as a catalytic RNA (1). RNAs need to be precisely folded into specific three-dimensional (3D) structures to function in biological processes such as protein translation and splicing but appear highly susceptible to misfolding (2). Like many structured RNAs, the Tetrahymena ribozyme assembles via a variety of folding pathways, many of which end up in nonnative kinetic traps (3). Under standard conditions in vitro, a small fraction folds to the native (N) state within 1 min, while the remaining fraction forms a long-lived misfolded intermediate (M) that refolds to the N state on a time scale of hours (3–5). The structural mechanism of this conformational switch remains unknown.The N state comprises an intricate catalytic core involving numerous strands and helices that bind the substrates of the splicing reaction along with an extensive ring of peripheral elements that “locks in” this core (6). Biochemical data indicate that the secondary and tertiary structures of M bear a close resemblance to N. Nevertheless, it has remained paradoxical that the transition from M to N is so slow when the states are so structurally similar to each other. A potential resolution to the paradox comes from a proposal that M and N are topologically distinct, with at least one pair of strands crossed in the core (7). The transition from the M state to the N state then cannot occur through a fast local rearrangement because of stereochemical constraints but instead requires unfolding of the ribozyme’s peripheral regions to release constraints on movement of core elements. Rearrangements of relative positions of core strands and helices in this unlocked state lead to the native-like topology, after which refolding of the peripheral tertiary contacts locks in the N state (3, 7–10). While this topological model is compelling and consistent with a wealth of biochemical data, the 3D structure of M has not been resolved, and it remains unknown which, if any, of the possible strand topological crossings are present in M.Here, we performed single-particle cryogenic electron microscopy (cryo-EM) to obtain structures of the M and N states. The structures provide unequivocal evidence for the topological isomer model, explain a large body of prior biochemical data and, by pinpointing a specific topological crossing, suggest possible pathways for transition from M to N. Our results illustrate the growing ability of cryo-EM to reveal complex structural changes in RNA folding pathways.     

HBcrAg Predicts Hepatocellular Carcinoma Development in Chronic B Hepatitis Related Liver Cirrhosis Patients Undergoing Long-Term Effective Anti-Viral

Hepatitis B core-related antigen (HBcrAg) is a predictor of hepatocellular carcinoma (HCC) in chronic hepatitis B (CHB) patients. Studies on anti-viral therapy have shown that the use of NUC therapy in HBV patients could reduce the incidence of HCC. However, the incidence of HCC continues to increase after long-term anti-viral therapy. The relationship between HBcrAg and HCC development in CHB-related liver cirrhosis (LC) patients undergoing long-term anti-viral therapy is still unclear. This study enrolled 1108 treatment-naïve CHB patients diagnosed with HBV-related LC receiving NUC therapy from April 1999 to February 2015. The baseline biomarkers, disease history, and following results were collected by the hospital. Among the 1108 patients, 219 developed HCC within a median follow-up period of 6.85 years. A multivariable Cox regression model was used, with adjustment for age, gender, FIB-4, DM, and HBsAg-HQ. The adjusted hazard ratios for the HBcrAg tertile levels were 1.70 (95%CI: 1.21, 2.39) and 2.14 (95%CI: 1.50, 3.05) for levels 3.4–4.9 and >4.9 logU/mL, respectively, compared with levels ≤3.4. The effect of the HBcrAg level on HCC incidence was found to be significantly modified by HBsAg-HQ, where lower HBsAg-HQ (≤ 3) values were associated with a significantly higher risk, but HBsAg-HQ levels >3 were not. Our results highlight that, after adjustment for potential confounding factors, patients with CHB-related LC and higher HBcrAg levels are at significant risk for HCC development, even while undergoing long-term effective anti-viral therapy. The HBcrAg level is therefore an independent risk factor for HCC development, especially for patients with HBsAg-HQ levels <3.     

Immune regulation by low doses of the DNA methyltransferase inhibitor 5-azacitidine in common human epithelial cancers

Epigenetic therapy is emerging as a potential therapy for solid tumors. To investigate its mechanism of action, we performed integrative expression and methylation analysis of 63 cancer cell lines (breast, colorectal, and ovarian) after treatment with the DNA methyltransferase inhibitor 5-azacitidine (AZA). Gene Set Enrichment Analysis demonstrated significant enrichment for immunomodulatory pathways in all three cancers (14.4-31.3%) including interferon signaling, antigen processing and presentation, and cytokines/chemokines. Strong upregulation of cancer testis antigens was also observed. An AZA IMmune gene set (AIMs) derived from the union of these immunomodulatory pathway genes classified primary tumors from all three types into “high” and “low” AIM gene expression subsets in tumor expression data from both TCGA and GEO. Samples from selected patient biopsies showed upregulation of AIM genes after treatment with epigenetic therapy. These results point to a broad immune stimulatory role for DNA demethylating drugs in multiple cancers.     

A quasi-experimental evaluation of advance care planning improves consistency between elderly individuals and their surrogates regarding end-of-life care preferences: Development and application of a decision aid with cartoon pictures

ObjectiveThis study sought to develop a decision aid with cartoon pictures and evaluate its effectiveness in increasing consistency between elderly individuals and their surrogates regarding end-of-life care.MethodsA pre-post quasi-experimental design was adopted using the Life Support Preferences Questionnaire. The intervention had two components: (1) increasing participants’ knowledge of medical treatments related to end-of-life care, and (2) sharing their end-of-life wishes. The experimental group received an intervention, whereas the control group received usual care.ResultsA total of 110 participants in 55 pairs of elderly individuals with average aged 86.4 and their surrogates (27 in the experimental group, 28 in the control group) were recruited from a veterans hospital in northern Taiwan. Nearly 90 % of elderly individuals were male. The multiple linear regression showed that the inconsistent gap between elderly individuals and their surrogates in the experimental group decreased 12 points than the control group after controlling the covariances (B = ?12.116, p = 0.032).ConclusionThe intervention improved the consistency between elderly individuals and their surrogates regarding end-of-life care.Practice implicationsA decision aid with cartoon pictures may support the discussion of end-of-life care in older Asian populations.