CHCHD2 maintains mitochondrial contact site and cristae organizing system stability and protects against mitochondrial dysfunction in an experimental model of Parkinson's disease

Background:Parkinson''s disease (PD) is the second most common neurodegenerative disease after Alzheimer''s dementia. Mitochondrial dysfunction is involved in the pathology of PD. Coiled-coil-helix-coiled-coil-helix domain-containing 2 (CHCHD2) was identified as associated with autosomal dominant PD. However, the mechanism of CHCHD2 in PD remains unclear.Methods:Short hairpin RNA (ShRNA)-mediated CHCHD2 knockdown or lentivirus-mediated CHCHD2 overexpression was performed to investigate the impact of CHCHD2 on mitochondrial morphology and function in neuronal tumor cell lines represented with human neuroblastoma (SHSY5Y) and HeLa cells. Blue-native polyacrylamide gel electrophoresis (PAGE) and two-dimensional sodium dodecyl sulfate-PAGE analysis were used to illustrate the role of CHCHD2 in mitochondrial contact site and cristae organizing system (MICOS). Co-immunoprecipitation and immunoblotting were used to address the interaction between CHCHD2 and Mic10. Serotype injection of adeno-associated vector-mediated CHCHD2 and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration were used to examine the influence of CHCHD2 in vivo.Results:We found that the overexpression of CHCHD2 can protect against methyl-4-phenylpyridinium (MPP+)-induced mitochondrial dysfunction and inhibit the loss of dopaminergic neurons in the MPTP-induced mouse model. Furthermore, we identified that CHCHD2 interacted with Mic10, and overexpression of CHCHD2 can protect against MPP⁺-induced MICOS impairment, while knockdown of CHCHD2 impaired the stability of MICOS.Conclusion:This study indicated that CHCHD2 could interact with Mic10 and maintain the stability of the MICOS complex, which contributes to protecting mitochondrial function in PD.     

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.     

The performance of five models compared with atrial high rate episodes predicts new atrial fibrillation after cardiac implantable electronic devices implantation

AimsSeveral predicting models have been evaluated for new‐onset atrial fibrillation (AF) in several clinical conditions, but never in patients with cardiac implantable electronic devices (CIED). We aimed to evaluate the five predicting models compared with atrial high rate episodes (AHRE) to predict new AF in patients with CIED.Methods and ResultsWe retrospective enrolled 470 consecutive patients with CIED and without a history of AF. The five predicting models, including CHA₂DS₂‐VASc score, C₂HEST score, mCHEST score, HAT₂CH₂ score, and HAVOC score were used. The primary endpoint was new AF documented by 12‐lead electrocardiography (ECG) or 30‐s ECG strip. Multivariable Cox regression analysis was used to determine variables associated with independent factors of new AF. Patients'' median age was 76 years and 58.7% were male. During follow‐up (median 29 months), 34 new AF occurred (incidence rate 2.99/100 patient‐years, 95% CI 1.67–6.20). Multivariable Cox regression analysis showed AHRE ≥6 min and 24 h, and HAT₂CH₂ score were independent predictors for new AF. Optimal AHRE cutoff value was 9.3 min with highest Youden index (AUC, 0.806; 95% CI, 0.722–0.889; p < .001). The AF occurrence rate of AHRE ≥9.3 min was 7 times AHRE <9.3 min (p < .001).ConclusionsWe compared 5 predicting models for new AF in patients with CIED and without a history of AF. AHRE ≥6 min and 24 h, and HAT₂CH₂ score were independent predictors for AF.     

Liver Function Derangement in Patients with Severe Fever and Thrombocytopenia Syndrome

Background and AimsPatients with severe fever with thrombocytopenia syndrome (SFTS) commonly show liver function impairment. This study aimed to characterize the liver function indices in SFTS patients and investigate their association with mortality.MethodsClinical information and laboratory results of 459 laboratory-confirmed SFTS patients, including 78 deceased and 381 surviving patients, were retrospectively analyzed. To explore the infectivity of SFTS caused by novel Bunyavirus (SFTSV) in hepatocytes, Huh7 human hepatoma cells were infected with various concentrations of SFTSV in vitro.ResultsThe proportion of SFTS patients developing liver injury during hospitalization was 73.2% (336/459); the hepatocellular injury was the predominant type. The median time to occurrence of liver injury from disease onset was 8 d. Liver injury in the deceased group occurred earlier than that in the surviving group. Alanine aminotransferase (ALT) level between 2–5 times upper limit of normal (ULN) at 4–6 d and between 5–15 ULN at 7–12 d of disease course were independent predictors of mortality. Alkaline phosphatase (ALP) >2 ULN at 7–9 d and elevated ALP at 10–12 days after disease onset were risk factors for death. ALT and aspartate transaminase (AST) levels were correlated with lymphocyte count and platelet-to-lymphocyte ratio (PLR). Total bilirubin (TB), ALT, AST levels showed positive correlation with viral load. In the in vitro experiment, SFTSV infected and replicated inside Huh7 cells.ConclusionsLiver injury is common in SFTS patients. ALT and ALP were independent predictors of SFTS-related mortality. Frequent monitoring and evaluation of liver function indices are needed for SFTS patients.     

Evaluation of a multiplex PCR kit for detection of 17 respiratory pathogens in hospitalized patients

BackgroundRapid pathogen identification is critical for optimizing diagnosis and treatment of infectious diseases. Multiplex polymerase chain reaction (PCR) is a sensitive, broad-spectrum molecular detection technique that is simple and rapid to perform. It is capable of simultaneously screening for multiple pathogens within a short time range. Here, we designed and evaluated a multiplex PCR kit for the identification of 17 common respiratory pathogens in clinical samples from hospitalized patients.MethodsA total of 452 samples from hospitalized patients, including 242 respiratory and 210 non-respiratory samples, were analyzed for 13 bacteria and 4 fungi by a multiplex fluorescent PCR kit. The diagnostic performance of the kit was assessed by considering routine microbiology as the reference standard.ResultsThe overall positivity rate of the multiplex PCR kit was 86.9%, much higher than that noted on routine microbiology (56.9%). Furthermore, the co-infection detection rate was also significantly higher than that noted on routine microbiology (69.5% vs. 15.0%). Compared with routine microbiology, kit sensitivity was >90% for detection of most target bacteria, with a negative predictive value (NPV) of >99%, especially for detection of Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, Stenotrophomonas maltophilia, Enterococcus faecium, Enterococcus faecalis and Escherichia coli. The kit was noted to be particularly superior in identifying Stenotrophomonas maltophilia and Streptococcus pneumoniae as compared to routine microbiology. The multiplex PCR kit was noted to be less sensitive (33.3–59.6%) and more specific (93.9–100.0%) for detection of mycobacteria and fungi.ConclusionsOur multiplex PCR kit offers a rapid and sensitive diagnosis of common bacterial pneumonia, although sensitivity for mycobacteria and fungi warrants enhancement. Further optimization includes minimizing false positivity and increasing relevance to clinical application.     

Preliminary validation study of the WHO quality of life (WHOQOL) scales for people with spinal cord injury in Mainland China

ObjectiveTo validate the WHOQOL Scales (WHOQOL-BREF and WHOQOL-DIS module) for people with spinal cord injury in Mainland China.DesignCross-sectional study.SettingShanghai Sunshine Rehabilitation Center.Participants249 adults with SCI who were admitted to a rehabilitation training program between 2017 and 2019.InterventionsNot applicable.MethodsQuestionnaires about personal and injury characteristics, the WHOQOL Scales, global QOL, Zung Self-Rating Anxiety/Depression Scale (SAS/SDS), and Community Integration Questionnaire (CIQ) were administrated. Floor and ceiling effects, reliability, and validity analyses were tested.ResultsThe 8 domains of the WHOQOL Scales showed no floor or ceiling effects. Cronbach alpha values of the WHOQOL-BREF and the WHOQOL-DIS were 0.93 and 0.78, respectively. Test-retest reliability was good for the WHOQOL Scales. Satisfactory criterion-related validity was shown by the correlation analysis among the WHOQOL Scales, SAS/SDS, CIQ, and global QOL. Good item-domain correlations (>0.50) were found for 38 items of the 39-item WHOQOL Scales, excepting the “impact of disability” (0.48) of the WHOQOL-DIS. Confirmatory Factor Analysis (CFA) supported a construct of the WHOQOL-DIS as made of four domains: autonomy, social inclusion, social activities, and discrimination. CFI and RMSEA values were 0.91 and 0.07, respectively, for the four-domain structure WHOQOL-DIS, with a higher-order factor. WHOQOL-BREF domains and WHOQOL-DIS scores showed the predicted pattern among a priori known groups.ConclusionThe WHOQOL Scales are valid and reliable, and they can be used to measure QOL in people with SCI in China. We suggest the WHOQOL-DIS be analyzed as one general item constituting a single 12-item domain.