Deconvolution methods infer quantitative cell type estimates from bulk measurement of mixed samples including blood and tissue. DNA methylation sequencing measures multiple CpGs per read, but few existing deconvolution methods leverage this within-read information. We develop CelFiE-ISH, which extends an existing method (CelFiE) to use within-read haplotype information. CelFiE-ISH outperforms CelFiE and other existing methods, achieving 30% better accuracy and more sensitive detection of rare cell types. We also demonstrate the importance of marker selection and of tailoring markers for haplotype-aware methods. While here we use gold-standard short-read sequencing data, haplotype-aware methods will be well-suited for long-read sequencing.

This study examined trajectories of repetitive sensorimotor (RSM), insistence on sameness (IS), and verbal RRBs from ages 2-19 in a well-characterized longitudinal cohort. We also tested the factor structure of the ADI-R restricted and repetitive behavior (RRB) domain at age 19 and the inclusion of a verbal RRBs factor, in addition to previously identified RSM and IS factors, at ages 9 and 19. The ADI-R was administered to 193 participants at five timepoints from 2 to 19. Confirmatory factor analysis (CFA) was used to examine the factor structure of ADI-R RRB data. Change in RRB subtypes was examined using group-based trajectory modeling and multilevel modeling. RSM and IS behaviors generally decreased from 2 to 19, with some participants experiencing increases from ages 2-9. 46.46% of participants experienced increasing verbal RRBs from 2 to 9, followed by a plateau from 9 to 19. The remaining participants had few to no verbal RRBs throughout development. Multilevel modeling indicated ADOS CSS scores were associated with change in RSM trajectories only. Child IQ was not associated with change in any RRB trajectories. CFA indicated previously-derived RSM and IS factors fit age 19 ADI-R data well. The inclusion of a third factor, verbal RRBs, worsened goodness-of-fit statistics and was ultimately omitted. Our findings suggest the RRB factor structure of the ADI remains consistent into early adulthood and illustrate developmental continuity and change in RRBs. For autistic individuals with fluent speech, RRB presentation may be influenced by verbal ability in ways that are highly variable, but clinically meaningful.

Regular exercise has many physical and brain health benefits, yet the molecular mechanisms mediating exercise effects across tissues remain poorly understood. Here we analyzed 400 high-quality DNA methylation, ATAC-seq, and RNA-seq datasets from eight tissues from control and endurance exercise-trained (EET) rats. Integration of baseline datasets mapped the gene location dependence of epigenetic control features and identified differing regulatory landscapes in each tissue. The transcriptional responses to 8 weeks of EET showed little overlap across tissues and predominantly comprised tissue-type enriched genes. We identified sex differences in the transcriptomic and epigenomic changes induced by EET. However, the sex-biased gene responses were linked to shared signaling pathways. We found that many G protein-coupled receptor-encoding genes are regulated by EET, suggesting a role for these receptors in mediating the molecular adaptations to training across tissues. Our findings provide new insights into the mechanisms underlying EET-induced health benefits across organs.

UNASSIGNED: DNA methylation is a crucial epigenetic alteration involved in diverse biological processes and diseases. Nevertheless, the precise role of DNA methylation in chemotherapeutic drug-induced alopecia remains unclear. This study examined the role and novel processes of DNA methylation in regulating of chemotherapeutic drug-induced alopecia.
UNASSIGNED: A mouse model of cyclophosphamide (CTX)-induced alopecia was established. Hematoxylin-eosin staining and immunohistochemical staining for the Ki67 proportion and a mitochondrial membrane potential assay (JC-1) were performed to assess the structural integrity and proliferative efficiency of the hair follicle stem cells (HFSCs). Immunofluorescence staining and real-time fluorescence quantitative PCR (RT-qPCR) were performed to determine the expression levels of key HFSC markers, namely Lgr5, CD49f, Sox9, CD200, and FZD10. Differential DNA methylation levels between the normal and CTX-induced model groups were determined through simple methylation sequencing and analyzed using bioinformatics tools. The expression levels of miR-365-1, apoptosis markers, and DAP3 were detected through RT-qPCR and western blotting. In parallel, primary mouse HFSCs were extracted and used as a cell model, which was constructed using 4-hydroperoxycyclophosphamide. The luciferase reporter gene assay was conducted to confirm miR-365-1 binding to DAP3. To measure the expression of relevant indicators, superoxide dismutase (SOD) and malondialdehyde (MDA) kits were used. Methylation-specific PCR (MS-PCR) was performed to determine DNA methylation levels. The regulatory relationship within HFSCs was confirmed through plasmid overexpression of miR-365-1 and DAP3.
UNASSIGNED: In the alopecia areata model, a substantial number of apoptotic cells were observed within the hair follicles on the mouse backs. Immunofluorescence staining revealed that the expression of HFSC markers significantly reduced in the CTX group. Both RT-qPCR and western blotting demonstrated a noteworthy difference in DNA methyltransferase expression. Simple methylation sequencing unveiled that DNA methylation substantially increased within the dorsal skin of the CTX group. Subsequent screening identified miR-365-1 as the most differentially expressed miRNA. miR-365-1 was predicted and confirmed to bind to the target gene DAP3. In the CTX group, SOD and ATP expression markedly reduced, whereas MDA levels were significantly elevated. Cellular investigations revealed 4-HC-induced cell cycle arrest and decreased expression of HFSC markers. MS-PCR indicated hypermethylation modification of miR-365-1 in the 4-HC-induced HFSCs. The luciferase reporter gene experiment confirmed the binding of miR-365-1 to the DAP3 promoter region. miR-365-1 overexpression dramatically reduced apoptotic protein expression in the HFSCs. However, this effect was slightly reversed after DAP3 overexpression in lentivirus.
UNASSIGNED: This study explored the occurrence of miR-365-1 DNA methylation in chemotherapeutic drug-induced alopecia. The results unveiled that miR-365-1 reduces cell apoptosis by targeting DAP3 in HFSCs, thereby revealing the role of DNA methylation of the miR-365-1 promoter in chemotherapeutic drug-induced alopecia.

DNA methylation, an epigenetic mechanism that alters gene expression without changing DNA sequence, is essential for organism development and key biological processes like genomic imprinting and X-chromosome inactivation. Despite tremendous efforts in DNA methylation research, accurate quantification of cytosine methylation remains a challenge. Here, a single-base methylation quantification approach is introduced by weighting methylation of consecutive CpG sites (Wemics) in genomic regions. Wemics quantification of DNA methylation better predicts its regulatory impact on gene transcription and identifies differentially methylated regions (DMRs) with more biological relevance. Most Wemics-quantified DMRs in lung cancer are epigenetically conserved and recurrently occurred in other primary cancers from The Cancer Genome Atlas (TCGA), and their aberrant alterations can serve as promising pan-cancer diagnostic markers. It is further revealed that these detected DMRs are enriched in transcription factor (TF) binding motifs, and methylation of these TF binding motifs and TF expression synergistically regulate target gene expression. Using Wemics on epigenomic-transcriptomic data from the large lung cancer cohort, a dozen novel genes with oncogenic potential are discovered that are upregulated by hypomethylation but overlooked by other quantification methods. These findings increase the understanding of the epigenetic mechanism by which DNA methylation regulates gene expression.

Aim: To explore the overall methylation changes in liver tissues during the formation of gallstones, as well as the key pathways and genes involved in the process. Methods: Reduced-representation bisulfite sequencing and RNA sequencing were conducted on the liver tissues of mice with gallstones and control normal mice. Results: A total of 8705 differentially methylated regions in CpG and 1410 differentially expressed genes were identified. The joint analysis indicated that aberrant DNA methylation may be associated with dysregulated gene expression in key pathways such as cholesterol metabolism and bile secretion. Conclusion: We propose for the first time that methylation changes in some key pathway genes in liver tissue may be involved in the formation of gallstones.

DNA methylation is an epigenetic modification that can alter gene expression, and the incidence can vary across developmental stages, inflammatory conditions, and sexes. The effects of viral maternal viral infection and sex on the DNA methylation patterns were studied in the hypothalamus of a pig model of immune activation during development. DNA methylation at single-base resolution in regions of high CpG density was measured on 24 individual hypothalamus samples using reduced representation bisulfite sequencing. Differential over- and under-methylated sites were identified and annotated to proximal genes and corresponding biological processes. A total of 120 sites were differentially methylated (FDR-adjusted p-value < 0.05) between maternal infection or sex groups. Among the 66 sites differentially methylated between groups exposed to inflammatory signals and control, most sites were over-methylated in the challenged group and included sites in the promoter regions of genes SIRT3 and NRBP1. Among the 54 differentially methylated sites between females and males, most sites were over-methylated in females and included sites in the promoter region of genes TNC and EIF4G1. The analysis of the genes proximal to the differentially methylated sites suggested that biological processes potentially impacted include immune response, neuron migration and ensheathment, peptide signaling, adaptive thermogenesis, and tissue development. These results suggest that translational studies should consider that the prolonged effect of maternal infection during gestation may be enacted through epigenetic regulatory mechanisms that may differ between sexes.

DNMT3A participates in de novo methylation, yet its impact on the proliferation of testicular Sertoli cells remains unclear. Development-specific methylation has been proven to be associated with cellular development. Therefore, in this study, we simulated DNMT3A expression pattern during testicular development by DNMT3A interference. Then, RRBS and RNA-seq were used to decipher DNMT3A regulatory mechanisms on Sertoli cell proliferation. Immunofluorescence staining revealed the expression of DNMT3A in the Sertoli cells of the prepubertal testis. DNMT3A was demonstrated to inhibit the cell cycle and proliferation of Sertoli cells, while promoting cell apoptosis. After transfected with DNMT3A interference, a total of 560 DEGs and 2,091 DMGs produced by DNMT3A interference were identified between two treated groups, respectively. Integrating the results from RRBS and RNA-seq, the overlapping genes between DMGs and DEGs were found to be enriched in the Gene Ontology (GO) terms related to cellular development and the Apelin signaling pathway. The present study demonstrated the impact of DNMT3A on the proliferation of porcine testicular Sertoli cells, suggesting that DNMT3A primarily acts through the Apelin signaling pathway. These findings provide valuable insights into how DNMT3A influences testicular development and health, offering new perspectives.

UNASSIGNED: Autistic females are frequently underdiagnosed, misdiagnosed, and/or diagnosed later in life. Restricted and Repetitive Behaviors (RRBs) are increasingly critical for diagnosis and yet are commonly rated lower in females. Whether this reflects genuinely lower levels of these traits, or if female-typical RRBs have a different phenotypic presentation that may not register on current quantitative measurement tools is unclear.
UNASSIGNED: Twenty-one autistic females and 21 autistic males matched on chronological age and FSIQ completed the AQ, ADOS-2, and ADI-R. Items from the ADOS-2 and ADI-R were selected that were relevant to the four areas of restricted and repetitive behavior in the DSM-5 diagnostic criteria of autism. Using a mixed-methods analytical approach, scores and comments on these measures were compared between sexes to better characterize RRBs in autistic females.
UNASSIGNED: There were no sex differences on the AQ, which broadly assesses autistic traits. When analyzed by the four DSM-5 RRB criteria, there were no sex differences on the ADI-R when using traditional algorithm scoring that narrows questions down to those that are more sensitive and specific in capturing autism in research samples with a high proportion of males. When incorporating additional items relevant to the DSM-5 to identify sex changes in a broader pool of items, females scored higher on stereotyped movements and speech. Females also engaged in more sensory behaviors during the ADOS-2. Qualitative analyses indicated that females were more likely to engage in stereotyped body rocking and spinning, stereotyped behaviors when anxious, to show major reactions to changes, repetitive language including listing and counting, and sensory behaviors, especially in the tactile domain.
UNASSIGNED: Exploratory findings highlight sex differences in RRBs that may help enhance diagnostic clarity for females. Higher tactile sensory behaviors in females suggests there may be increased diagnostic sensitivity for females with the updated DSM-5 that now includes sensory components as part of the diagnostic criteria.

The central dogma of molecular biology dictates the general flow of molecular information from DNA that leads to a functional cellular outcome. In skeletal muscle fibers, the extent to which global myonuclear transcriptional alterations, accounting for epigenetic and post-transcriptional influences, contribute to an adaptive stress response is not clearly defined. In this investigation, we leveraged an integrated analysis of the myonucleus-specific DNA methylome and transcriptome, as well as myonuclear small RNA profiling to molecularly define the early phase of skeletal muscle fiber hypertrophy. The analysis of myonucleus-specific mature microRNA and other small RNA species provides new directions for exploring muscle adaptation and complemented the methylation and transcriptional information. Our integrated multi-omics interrogation revealed a coordinated myonuclear molecular landscape during muscle loading that coincides with an acute and rapid reduction of oxidative metabolism. This response may favor a biosynthesis-oriented metabolic program that supports rapid hypertrophic growth.