Epigenomics

Plac1 is an X-linked gene essential for placental and embryonic development. A knockout (KO) mouse model was used to identify Plac1-regulated gene expression at E16.5 and E18.5 using gene expression microarray. Genes exhibiting at least 1.5-fold change in expression and FDR < .05 were considered significant. At E16.5, 717 genes were downregulated and 798 were upregulated in male KO placentas versus wild type (WT), whereas at E18.5, 1122 genes were downregulated and 1149 were upregulated. GO, KEGG, and IPA analyses revealed downregulated genes were enriched for Rho GTPase-mediated and actin-cytoskeleton based processes that transmit extracellular cues through canonical signaling pathways, including Integrin, GPCR, Wnt, Notch, VEGF, BMP and TGF-beta, documented to impact trophoblast development, vasculogenesis, vascular tone, branching morphogenesis, and immunomodulation. Furthermore, a preeclampsia-associated transcriptomic signature was induced that strengthened over time. By contrast, upregulated genes reflected immune activation and adaptations to oxidative stress resulting from impaired placental function. These findings indicate that Plac1 supports signaling required to maintain placental structure and regulatory function. Its absence disrupts essential regulatory processes and triggers cellular stress and immune activation, contributing to fetal growth restriction, increased risk for embryopathy and preeclampsia, consistent with the Developmental Origins of Health and Disease (DOHaD) framework.

BackgroundHypertension and preeclampsia are clinically distinct, yet biologically related conditions characterized by vascular dysfunction and elevated cardiovascular risk. Although genome-wide association studies (GWAS) have identified loci associated with blood pressure traits and preeclampsia, the functional mechanisms linking shared variants to gene regulation and clinical phenotypes remain unclear. MethodsWe integrated GWAS summary statistics for hypertension, systolic blood pressure (SBP), diastolic blood pressure (DBP), and preeclampsia to identify shared variants (p [&le;] 1x10-). Cis-expression quantitative trait loci (eQTL) analyses were performed in whole blood using RNA-seq data from 180 African American women. Significant associations (FDR [&le;] 0.05) were evaluated for replication across vascular, metabolic, and endocrine tissues in the Genotype-Tissue Expression (GTEx) project. Associations between gene expression and blood pressure traits were also assessed. ResultsWe identified 4,792 shared GWAS variants, of which 4,663 were tested in eQTL analyses, yielding 1,837 significant variant-gene associations across 78 genes. Replication in GTEx confirmed 645 associations involving 24 genes, many showing cross-tissue regulatory effects. Three genes (C4B, HLA-C, and HLA-DQB1) demonstrated convergent evidence across GWAS, gene regulation, and expression-trait analyses. C4B expression was positively associated with hypertension and SBP, while HLA-C showed consistent negative associations with hypertension, SBP, and DBP. HLA-DQB1 expression was specifically associated with DBP, suggesting trait-specific effects. ConclusionsThese findings highlight immune-related pathways as key mediators linking hypertension and preeclampsia. Integrating genetic, transcriptomic, and phenotypic data provides a framework for identifying functionally relevant loci and advancing mechanistic insights into cardiometabolic and pregnancy-related disorders. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=116 SRC="FIGDIR/small/26352450v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@1332b09org.highwire.dtl.DTLVardef@4e7c49org.highwire.dtl.DTLVardef@c1b980org.highwire.dtl.DTLVardef@799767_HPS_FORMAT_FIGEXP M_FIG C_FIG Shared genetic variants across hypertension, blood pressure traits, and preeclampsia converge on immune regulatory genes linking gene regulation to clinical phenotypes. GWAS summary statistics for hypertension, SBP, DBP, and preeclampsia were intersected to identify 4,792 shared variants, of which 4,663 were tested in cis-eQTL analyses in whole blood from 180 African American women (left). Shared variants regulate immune-related genes through cis-eQTL effects, yielding 1,837 associations involving 78 genes (FDR [&le;] 0.05). Three convergent genes emerged: C4B (upregulated), HLA-C (downregulated), and HLA-DQB1 (upregulated), with 645 associations involving 24 genes replicated across eight tissues in GTEx (center). Expression-trait analyses confirmed that C4B expression was positively associated with hypertension and SBP, HLA-C expression was negatively associated with hypertension, SBP, and DBP, and HLA-DQB1 expression was specifically associated with DBP. These genes implicate complement activation, antigen presentation, and adaptive immunity as shared mechanisms contributing to vascular dysfunction in both hypertension and preeclampsia. eQTL indicates expression quantitative trait locus; FDR, false discovery rate; GTEx, Genotype-Tissue Expression project; SBP, systolic blood pressure; DBP, diastolic blood pressure; APC, antigen-presenting cell; TCR, T-cell receptor; MHC, major histocompatibility complex.

The eukaryotic ribosomal genes are multi-copy genes, transcribed from the rDNA, and approximately one third of them is actively transcribed in differentiated cells. A number of lncRNAs have been identified from the intergenic spacer between the rRNA genes, among those the spacer RNA and PAPAS that are involved silencing of rRNA gene copies by altering the chromatin configuration. Here, we have identified lncRNAs that are transcribed from the human rDNA loci and modulate the loci; IGS38 positively regulates rRNA gene transcription by associating to the 47S rRNA gene promoter and modulating the rRNA promoter accessibility while IGS32as associates with heterochromatin. IGS38 binds to the 47S gene promoter through the RNA pol I factors TAF1C and RRN3 as well as the Williams Syndrome Transcription Factor (WSTF), a component of the B-WICH chromatin remodelling complex. The increased accessibility of the promoter stabilises the architectural protein Upstream Binding Factor (UBF) at the rRNA promoter, thereby facilitating RNA pol I promoter escape. Furthermore, IGS38 knock down displays and increased dsRNA abundance in the cytoplasm with a weak induction of the dsRNA sensor OAS2, typically induced by interferon and viral dsRNA. Overall, the both IGS38 and IGS32as are chromatin associated lncRNAs involved in rDNA chromatin changes, and IGS38 is stimulating, together with WSTF, rRNA gene transcription in human cells. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=199 HEIGHT=200 SRC="FIGDIR/small/722362v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@14d4159org.highwire.dtl.DTLVardef@fd773forg.highwire.dtl.DTLVardef@a0030dorg.highwire.dtl.DTLVardef@1285301_HPS_FORMAT_FIGEXP M_FIG C_FIG IGS stabilises 47S rRNA transcription, disruption of IGS38 expression leads to the release of dsRNA in the cytoplasm and a weak immune activation of OAS2. Created by biorender (https://biorender.com/shortURL)

Enhancers are key epigenetic regulatory elements that orchestrate spatiotemporal gene expression and are critical in mammalian development, gene regulation, and disease. Histone modifications such as H3K4me1 (a canonical enhancer mark) and H3K27ac (which distinguishes active enhancers) remain poorly characterized during early mammalian embryogenesis. Using low-input CUT&RUN (Cleavage Under Targets and Release Using Nuclease) with input as low as 50 cells, this study profiles genome-wide H3K4me1 and H3K27ac patterns in mouse oocytes and pre-implantation embryos. Both marks are enriched in distal regions and exhibit distinct sequence preferences and reprogramming dynamics in pre-implantation embryos. H3K27ac is reprogrammed at the 2-cell stage and marks active enhancers, while H3K4me1 is remodeled at the 4-cell stage and co-localizes with H3K27ac, overlapping with accessible chromatin regions. Interestingly, the co-localization of H3K4me1 and H3K27ac is also detected in promoter regions, where they exhibit a mutually exclusive pattern with H3K4me3. Three enhancer types-active (H3K4me1/H3K27ac), primed (H3K4me1), and poised (H3K4me1/H3K27me3)-are dynamically remodeled during maternal-to-zygotic transition (MZT), with active enhancers increasing significantly after zygotic genome activation. Furthermore, genome-wide super-enhancers are identified and mainly enriched in promoters. The differences in gene expression at different stages may be related to the specific motifs enriched by super-enhancers.

Objective: To characterize genome-wide DNA methylation patterns associated with sepsis using the Infinium Methylation EPIC v2.0 platform and to evaluate the feasibility of pooled methylation profiling in a pilot critical care cohort. Design: Single-center pilot epigenome-wide association study using pooled whole-blood genomic DNA and pool-level bioinformatic analysis. Setting: Academic medical center. Patients: Fifty critically ill adults enrolled within 48 hours of illness onset and 20 healthy controls. Interventions: None. Measurements and Main Results: Critically ill patients required mechanical ventilation and/or vasopressor support. Sepsis was defined according to Sepsis-3 criteria. Seventy individual samples were organized into 14 intended pools of 5 individuals each: 7 sepsis pools, 3 critically ill non-septic pools, and 4 healthy-control pools. One critically ill non-septic pool was excluded because of poor DNA quality, yielding 13 analyzable pools. For the primary pooled comparison, 7 sepsis pools were compared with 6 non-sepsis comparator pools comprising 2 critically ill non-septic and 4 healthy-control pools. After quality control and preprocessing with SeSAMe, 876,094 CpG sites were retained. The initial pool-level screen identified 170,897 candidate differentially methylated regions. Application of stringent secondary filters (false discovery rate <= 1%, absolute delta-beta >= 7.5%, and >= 5 CpGs per region) yielded a high-confidence subset with marked directional skewing, including 155 hypomethylated and 32 hypermethylated regions in sepsis. Differentially methylated region-associated genes were enriched in myeloid leukocyte activation, myeloid leukocyte-mediated immunity, defense response to bacterium, neutrophil granule biology, and hematopoietic cell lineage pathways. Additional signals involved microRNA-associated targets, ribosome biogenesis, RNA processing, long noncoding RNAs, and previously uncharacterized loci. Conclusions: In this pilot pooled EPIC v2.0 study, sepsis was associated with a biologically coherent, predominantly hypomethylated methylation signature enriched in myeloid and host-defense pathways. These findings support the feasibility of pooled methylation profiling for discovery-oriented sepsis biobank studies but should be interpreted as hypothesis-generating given the pool-level design, limited effective sample size, heterogeneous comparator group, and lack of direct validation against individual-level methylation profiles.

Congenital heart defects (CHDs) are the most common congenital malformations and often arise from perturbations during early embryonic development. Maternal metabolic disturbances in early pregnancy may contribute to CHD risk, but evidence from early first-trimester metabolomics studies is limited. We conducted an untargeted metabolomics case-control study using early first-trimester maternal plasma samples (gestational weeks 4-10) from the Danish National Birth Cohort. Metabolite profiling was performed via liquid chromatography-tandem mass spectrometry (LC-MS/MS) on 160 matched CHD case-control pairs (320 total samples). Conditional logistic regression and interaction analysis were used to identify metabolites associated with CHD risk or specific cardiac phenotypes. A total of 1,471 metabolite features were measured with 69 metabolites being associated with CHD at nominal significance (p < 0.05). These included a desaturated analog of sphingosine-1-phosphate (S1P), isoleucylproline and an arginine related metabolite. However, after false discovery rate correction for multiple testing no metabolites remained significant. While these findings do not preclude that subtle metabolic variation may exist in early pregnancy among CHD cases, they also underscore the challenges of biomarker discovery in this context. This work highlights the potential of early-pregnancy metabolomics for CHD biomarker discovery, and points toward more targeted future studies with improved sample collection protocols, pre-specified pathway panels, and phenotype-homogeneous analyses to better capture the subtle metabolic variation that may underlie CHD risk.

Background: Hypertensive disorders of pregnancy contribute substantially to maternal morbidity and mortality, and occur with increased frequency among women with uterine fibroids. Biomarkers involved in oxidative stress and endothelial function, including folate, vitamin B12, vitamin D, and homocysteine, have been studied in relation to hypertensive disorders of pregnancy, but their relationship to fibroid-associated risk has not been well characterized, particularly in racially and ethnically diverse populations. Study Design: This study was a retrospective analysis of the Boston Birth Cohort, a prospective cohort recruited at a large urban medical center. The analytic sample included 722 women with complete data on hypertensive disorder status, uterine fibroid status, and plasma biomarker measurements. Uterine fibroids and hypertensive disorders of pregnancy were ascertained through physician-assigned diagnostic codes and ultrasound report review. Plasma folate, vitamin B12, vitamin D, and homocysteine were measured in maternal or cord blood and analyzed as continuous variables and quartiles. Multivariable logistic regression models were used to estimate independent associations, evaluate interaction terms, and assess joint exposure categories. Results: Of the 722 participants, 12% (86/722) had uterine fibroids and 10% (72/722) had a hypertensive disorder of pregnancy. Plasma micronutrient concentrations did not differ significantly by fibroid status. Women with hypertensive disorders of pregnancy had higher plasma homocysteine concentrations compared with those without (p=0.028). Hypertensive disorders of pregnancy were more common in the lowest folate quartile compared with the highest quartile (p=0.018) and in the highest homocysteine quartile compared with lower quartiles (p=0.031). In joint-effects analyses, higher odds of having a hypertensive disorder of pregnancy were observed among women with both uterine fibroids and low folate compared with women without fibroids and with adequate folate (p=0.027). No significant joint associations were observed for vitamin D, vitamin B12, or homocysteine. Conclusion: In this cohort, the co-occurrence of uterine fibroids and lower folate concentrations was associated with hypertensive disorders of pregnancy. This joint exposure delineates a subgroup that may be clinically relevant for future studies aimed at refining maternal risk characterization and exploring targeted nutritional supplementation strategies.

Background Liver fibrosis (LF) represents a pivotal pathological phase in the advancement of chronic liver disorders toward cirrhosis. Amino acid metabolism reprogramming plays a pivotal role in its pathogenesis, yet the underlying molecular mechanisms remain incompletely understood. Methods Integrating three public datasets (GSE14323, GSE84044, and GSE136103) with amino acid metabolism-related gene sets, we performed consensus clustering, machine learning algorithms, functional enrichment analysis, immune microenvironment composition, regulatory network construction, and drug prediction. Results Fibrotic samples were classified into two amino acid metabolism-related subtypes with distinct immune landscapes and functional phenotypes. Through integrated analysis of differentially expressed genes (DEGs) common to both subtypes, fibrotic versus control comparisons, and amino acid metabolism-related gene sets, four biomarkers, GSTP1, LDHB, OXCT1, and PTGDS, were identified. These biomarkers were enriched in pathways related to epithelial-mesenchymal transition, interferon responses, and TNF/NF-{kappa}B signaling. Notably, GSTP1 and LDHB positively correlated with M1 macrophage infiltration and negatively with regulatory T cell abundance. Single-cell transcriptomic analysis revealed that cholangiocytes expressed all four biomarkers with elevated levels in fibrosis and interacted with macrophages/mesenchymal cells via MIF-CD74/CXCR4. Regulatory network analysis highlighted key modulators, including MALAT1, hsa-miR-3163, OXCT1, SMAD4, and RELA. Furthermore, 5-fluorouracil was predicted as a multi-target compound, with the strongest predicted binding affinity for OXCT1. In vitro validation confirmed the upregulation of GSTP1 and LDHB, aligning with the bioinformatics findings. Conclusion This study identified four amino acid metabolism-related biomarkers, revealing immune heterogeneity and cholangiocyte-centered intercellular communication in LF. These findings establish a foundation for biomarker-based diagnosis, subtype-guided patient stratification, and the development of cell-type-specific therapeutic strategies in LF.

Background Preterm birth (PTB) rates among Hispanic/Latina individuals in the United States have risen over the past decade. Data suggests this rise may be driven in part by psychosocial stress. Leukocyte telomere length (LTL), a marker of cumulative cellular aging that shortens under chronic stress, may capture stress-related biological vulnerability, but has not been examined as a potential population-level contributor to PTB in Hispanic/Latina pregnancies. Objective To examine the association between mid-pregnancy maternal LTL and PTB in a population-based Hispanic/Latina cohort. Methods In a case-control study nested within a California singleton birth cohort (n = 436 Hispanic/Latina individuals; 215 PTB, 221 term births), LTL was measured by quantitative PCR from biobank specimens collected from 15 to 20 weeks of gestation. Covariates from linked birth certificate and hospital discharge records were included. Logistic regression estimated ORs and 95% CIs of PTB by LTL examined continuously and by percentile category (<=10th, 11th-89th, >=90th) with and without adjustment for covariates. Results Mean and median LTL did not differ between PTB and term births. LTL at or below the 10th percentile was associated with elevated odds of PTB relative to full-term birth (12.6% versus 4.3%; ORc = 3.2, 95% CI 1.3-7.9), persisting after partial (ORadj1 = 3.2, 95% CI 1.3-8.3) and full covariate adjustment (ORadj2 = 3.4, 95% CI 1.3-9.3). Subgroup analyses showed consistent directional patterns across PTB subgroups and for early term birth (ORadj2 = 5.1, 95% CI 1.5-17.0). Conclusions Mid-pregnancy maternal LTL <=10th percentile was associated with more than three times the odds of PTB, with risk concentrated at the extreme low tail of the distribution. Consistent with a cumulative allostatic load model, markedly short LTL at mid-gestation may reflect elevated stress-related biological risk for preterm delivery. These findings support upstream investment in stress reduction and prospective LTL research in high-burden populations.

Cartilage and bone that comprise craniofacial structures as well as neurons and glia of the peripheral nervous system are derived from a multipotent population of cranial neural crest cells, that respond to both cell intrinsic and extrinsic cues to differentiate into precise cell states. Both a genetic and epigenetic regulatory network are required for each step in the differentiation process, involving transcription factors, histone modifiers and chromatin remodelers. Here, we examined the direct transcriptional targets of two histone methyltransferases, Prdm3 and Prdm16 in zebrafish neural crest cells at 48 hours post fertilization in zebrafish. Using CUT&RUN, we examined both direct DNA binding and nucleosome association. At this stage of development, CUT&RUN fragment size analysis indicated that Prdm3 and Prdm16 are largely associated with nucleosomes. We further analyzed these nucleosome peak sets to identify 6 clusters where differential binding of Prdm3 and Prdm16 and differential enrichment of gene ontology terms for target genes was observed. We validated gene expression in each cluster by in situ hybridization chain reaction (HCR) at 48 hpf demonstrating that prdm3 and prdm16 mutants exhibit corresponding changes in gene expression of the putative gene targets identified. Finally, we performed CUT&RUN-qPCR in prdm3 and prdm16 mutant zebrafish embryos and demonstrated reduced binding at putative target loci. Together these data suggest that Prdm3 and Prdm16 regulate their transcriptional targets primarily by binding nucleosomes around their putative target loci to control downstream gene expression. HighlightsPrdm3 and Prdm16 associate with nucleosomes for regulation of gene expression Gene targets are altered in prdm3 and prdm16 mutant zebrafish Reduced binding is observed in respective mutants

Platinum resistance remains a major barrier in Ovarian cancer (OC) treatment[1]. While hyperactivation of DNA damage response (DDR) is a hallmark of chemoresistance[2], the underlying epigenetic mechanisms driving this adaptation remain poorly understood. Here, we identify a novel post-transcriptional regulatory axis involving miR-221-5p that governs two critical DDR effectors: RAD18, which mediates DNA damage tolerance through trans-lesion synthesis (TLS)[3][4], and RAD51, the central recombinase for homologous recombination (HR)[5][6]. Although the miR-221/222 cluster is traditionally categorized as oncogenic[7][8], we demonstrate that the miR-221-5p arm functions as a potent tumor suppressor in OC. Bioinformatic and luciferase reporter assays confirmed that miR-221-5p directly targets the 3'UTRs of both RAD18 and RAD51. In OC clinical specimens and cell lines, miR-221-5p downregulation inversely correlates with RAD18/RAD51 expression. Functionally, miR-221-5p restoration suppressed platinum-induced PCNA mono-ubiquitination and HR, inducing a "functional BRCAness" that sensitized both established and patient-derived primary OC cells to carboplatin and PARP inhibition. Furthermore, in vivo disseminated xenograft models demonstrated that stable miR-221-5p expression significantly reduced tumor burden. Collectively, our results delineate a novel regulatory mechanism where loss of miR-221-5p drives chemoresistance by derepressing the RAD18/RAD51 axis, identifying this axis as a promising therapeutic target.

Emerging evidence indicates that the maternal in utero environment has enduring effects on offspring neurodevelopment. The obesity epidemic in the United States affects nearly one-third of women before pregnancy, potentially predisposing offspring to harmful developmental conditions. Glucose, the primary energy source for the brain, is highly regulated by facilitative diffusion glucose transporters (GLUTs). However, our understanding of how maternal obesity influences perinatal cerebral glucose metabolism remains limited. We hypothesized that maternal obesity is associated with altered expression of key GLUTs and dysregulated energy-sensing mechanisms in the fetal brain. Female C57BL/6J mice were randomly assigned to either a control diet (CON) or an obesogenic diet (DIO) (60% kcal from fat, 17.5% kcal from sucrose) for 10 weeks, time-mated with control males, and fed their respective diets throughout gestation. At 18.5 days post coitum, fetal brain tissue was collected for protein analysis. DIO diet did not affect litter size, offspring body weight, or brain weight when compared to CON. Whole brain GLUT1 expression was elevated only in female DIO offspring, while GLUT3 and GLUT4 expression was increased in all DIO offspring without modification by sex. However, maternal diet was not associated with differences in the activation of energy regulatory pathways adenosine monophosphate-activated protein kinase (AMPK) or the nutrient-sensing pathway mechanistic target of rapamycin (mTOR) in the fetal brain. These findings suggest that maternal obesogenic diet alters glucose transporter expression in the fetal brain, indicating a potential disruption in cerebral glucose metabolism during critical periods of perinatal development.

Preterm birth is associated with alterations in early caregiver-infant regulation, with potential consequences for socio-emotional and physiological development. However, the mechanisms through which early interactional experience shapes these processes remain unclear. Here, we tested whether a structured dyadic intervention could modify co-regulatory dynamics across physiological, behavioral, and relational levels. Fifty-four 7-month-old preterm infants and their parents were assigned to either a shared book reading intervention (n = 22) or an active control condition based on a shared building activity (n = 32) and compared with 39 full-term infants. The intervention consisted of an 8-week program of shared book reading, designed to structure parent-infant interaction. Physiological synchrony was assessed at the dyadic level, alongside infants autonomic regulation and cardiovascular signal complexity. Behavioral engagement and parental attachment representations were also evaluated. Results showed that mother-infant physiological synchrony emerged selectively within the interactional context trained by the intervention and only in the intervention group. This context-specific synchrony was accompanied by modulation of vagal activity and increased cardiovascular complexity in preterm infants, consistent with enhanced flexibility of autonomic control. At the behavioral and relational levels, intervention infants showed increased initiating joint attention, while parents reported higher secure attachment. These findings support a model of experience-dependent early synchrony, in which repeated dyadic interaction through shared book reading shapes the coupling between interpersonal coordination and individual physiological regulation. By linking synchrony, autonomic flexibility, and social engagement, this study identifies a mechanism through which early caregiving experience can organize developmental trajectories following prematurity.

Vitamin D signaling has recognized roles in female reproductive physiology, but its effects at the chromatin level in endometrial stromal cells are still unclear. Here, we investigated how the active form of vitamin D, 1,25-dihydroxyvitamin D3, or calcitriol, influences the accessible chromatin landscape of human endometrial stromal cells. Assay for transposase-accessible chromatin using sequencing (ATAC-seq) was performed on T-HESCs treated with either a vehicle or 1,25(OH)2D3. Ligand treatment increased overall chromatin accessibility, shown by higher ATAC-seq signal intensity, while causing only minor changes in the total number of called peaks. Peak annotation revealed that accessible regions were spread across both promoter-proximal and distal genomic areas. Integrating this data with CUT&RUN and RNA sequencing showed that most vitamin D-responsive cistromic modifications and transcripts were linked to nearby open chromatin, though fewer were associated with regions that were significantly differentially accessible. These results suggest that 1,25(OH)2D3-dependent transcription mainly occurs within a permissive, pre-accessible chromatin environment. This study offers new evidence that active vitamin D influences the epigenomic landscape of human endometrial stromal cells, establishing the chromatin-based molecular response to a chemically-defined VDR ligand, 1,25(OH)2D3, relevant to stromal differentiation and preparation for decidualization. HighlightsO_LIFirst evidence suggesting the direct impact of active vitamin D, 1,25-dihydroxyvitamin D3, 1,25(OH)2D3, enhanced the signal intensity of chromatin accessibility in human endometrial stromal cells C_LIO_LIMost accessible chromatin regions were shared between vehicle and ligand-treated human endometrial stromal cells C_LIO_LI1,25(OH)2D3-responsive transcription occurs largely within pre-accessible chromatin in human endometrial stromal cells C_LIO_LIAssay for transposase-accessible chromatin sequencing (ATAC-seq) defines a chromatin-level pharmacologic response to a chemically defined VDR ligand in human endometrial stromal cells C_LI

Study questionDoes the human placenta utilize the creatine phosphagen system for energy homeostasis during development? Summary answerComponents of the creatine (Cr)-creatine kinase (CK)-phosphocreatine (PCr) system are dynamically expressed by the trophoblast and mesenchymal compartments throughout gestation wherein creatine kinase is required for cellular ATP metabolism, cell cycle, and proliferation of trophoblast cells. What is known alreadyThe Cr-CK-PCr system maintains ATP homeostasis in tissues with high energy demand and is required for proliferation, migration, and invasion of tumor cells. The term human placenta can synthesize and transport creatine locally. Early placental development involves trophoblast proliferation, an event requiring ATP, but the role of the creatine phosphagen system during early placental development remains unknown. Study design, size, durationWe performed immunohistochemistry (IHC) and immunofluorescence (IF) for different components (biosynthesis, transport, utilization) of the Cr-Ck-PCr system in human placentae (n=3/group) across gestation including first trimester, second trimester, and term. Using primary human trophoblast stem cells (hTSCs) and trophoblast organoids (TO), we determined the role of the creatine phosphagen system in trophoblast growth by functional inhibition of creatine kinase. Participants/materials, setting, methodsIHC/IF were performed in human placentae across gestation for proteins involved in biosynthesis (AGAT and GAMT), transport (SLC6A8, SLC22A15, and SLC6A13) and utilization (CKB and CKMT1) of creatine to determine the presence of the creatine phosphagen system locally in the placenta. For delineating the functional importance of this system in placental development, cyclocreatine (cCr), a creatine analogue, was used for functional inhibition of CK. Primary hTSCs were culture in medium containing 0 (control), 1, 10, 20 mM cCr for 48 hours followed by analysis of cell growth (cell count), cell cycle (EdU incorporation assay), apoptosis (Annexin V/PI flow cytometry), energy metabolism (Sea horse mito-stress and glycolytic stress tests), and gene expression (qPCR). Primary TO were also treated with 20mM cCr for 6 days in vitro to determine the role of Cr-CK-PCr system in placental development. Main results and the role of chanceAGAT localized to the fetal villous mesenchyme, while GAMT was broadly expressed in the trophoblast and fetal mesenchyme compartments across gestation. CKB localized primarily to fetal mesenchyme with strongest expression at term. CKMT1 was broadly expressed in all trophoblast subtypes. SLC6A8 was abundant in early syncytiotrophoblast but absent at term, where its expression shifted to fetal blood vessels. SLC22A15 was expressed in the endothelial cells of fetal capillaries across gestation. In primary hTSCs, cyclocreatine (20mM) treatment reduced proliferation (P<0.001), decreased expression of trophoblast epithelial marker EGFR (P<0.05), induced G0/G1 and G2/M arrests (P<0.0001), enhanced early and late apoptosis (P<0.0001), and downregulated GPX8 expression (P<0.05). Seahorse analysis revealed marked reductions (P<0.01) in mitochondrial (basal, maximal, and ATP-linked) and glycolytic (rate, capacity, and reserve) function compared to controls. In primary human TO, cyclocreatine treatment reduced the growth of organoids (P<0.05) as well the expression of EGFR (P<0.05). Large scale dataN/A Limitations, reasons for cautionFurther experiments assessing apoptosis, cellular stress and redox imbalance may provide more mechanistic role of the creatine phosphagen system in trophoblast metabolism and function. Since the functional role of the Cr-CK-PCr system was investigated in vitro, findings of this study should be taken with caution for implications of in vivo placental development. Nevertheless, reproducible results of reduced growth of trophoblast cells using both 2D and 3D cultures is highly suggestive of the importance of the creatine phosphagen system in early placental development. Wider implications of the findingsThis study provides foundational knowledge that the placenta contains the creatine phosphagen system, known for ATP homeostasis, and that this system ensures proper cell division, survival and placental development. Dysregulation of components of Cr-CK-PCr system in placenta has been observed in pregnancy disorders such as preeclampsia and fetal growth restriction warranting continued investigation into mechanisms and potential remediation using creatine supplementation. Stem cells share similar metabolic features so findings of this study can be implicated in other stem cells models as well. Study funding/competing interest(s)This work was supported by CIRM EDUC4-12804 Interdisciplinary Stem Cell Training Grant and a Lalor Foundation Postdoctoral Fellowship awarded to NS, and by the California Institute for Regenerative Medicine (DISC0-13757) and the National Institute of Child Health and Human Development (R01-HD096260) award to FS. The authors have no competing interest to declare.

BackgroundHypertension is a highly heritable cardiovascular disorder and a major determinant of cardiometabolic disease, including diabetes. However, the regulatory genes and tissue-specific mechanisms underlying blood pressure variations remain incompletely understood. MethodsLeveraging a well-characterized prospective population-based cohort comprised of 27,308 participants from the Singapore Chinese Health Study (SCHS), we evaluated genome-wide genetic associations for five blood pressure traits: hypertension status, systolic blood pressure, diastolic blood pressure, mean arterial pressure (MAP) and pulse pressure (PP). Additionally, we conducted a transcriptome-wide association study (TWAS), integrating gene expression data from 49 tissues, followed by colocalization and fine-mapping to prioritize regulatory genes. Association of identified variants with incident diabetes was additionally evaluated in the longitudinal data. ResultsWe validated 10 blood pressure loci (P between 1.64 x 10-20 - 4.10 x 10-8) and identified an East-Asian specific splice donor variant at the COL21A1 gene associated with PP (rs149344559, P = 6.78 x 10-10). Integrative analyses prioritized FGF5 in kidney cortex and ENPEP in pituitary tissue as candidate regulatory genes. The blood pressure-lowering allele at ENPEP (T allele, rs1879056) was associated with reduced risk of incident diabetes. Mediation analysis demonstrated that approximately 21% of the genetic association with diabetes was mediated through MAP (Pindirect-effect = 2 x 10-16). ConclusionThis study refines genetic predispositions for blood pressure among East-Asians. We further delineate tissue-specific regulatory pathways underlying blood pressure variations and identify ENPEP-mediated dysfunctions linking blood pressure genetics to diabetes risk, underscoring integrated disease mechanisms.

Tobacco smoking induces DNA methylation (DNAm) changes in blood and other tissues, which may influence chronic health outcomes. However, the breadth of smoking-related DNAm changes remains unmapped, offering a space for employing novel technologies. To expand our understanding of smoking impacts on DNAm, we conducted an epigenome-wide association study (EWAS) comparing ever smokers to never smokers, using blood from a multiethnic U.S. study population (n=887). We employed the newly developed Illumina Methylation Screening Array (MSA) covering 269,094 unique sites, including 123,776 CpGs not assayed in previous EWAS. Trans-ethnic meta-analysis identified 152 differentially methylated positions (DMPs) associated with ever-smoking status (n=764); European-specific analysis yielded 129 DMPs (n=674), including 106 overlapping with trans-ethnic analysis. A separate, large-scale replication EWAS (n=2,190) confirmed 91 trans-ethnic and 77 European-specific DMPs. Among our findings, we identified 61 DMPs at CpGs novel to the MSA platform, including near both new and known smoking-associated genes. Most notably, we uncovered a dense cluster of 12 DMPs within a 1117 bp region of ECEL1P1, forming the most long-lasting, persistent smoking-associated DMR ever detected, even among former smokers who quit decades prior. We also detected new signals at AHRR, a well-known locus for smoking-related DNAm changes. eFORGE analysis revealed that detected smoking-associated DNAm changes are predominantly located in hematopoietic stem and progenitor cell (HSPC) DNase I hotspots, aligning with gene set enrichment analyses that highlighted pathways related to hematopoietic stem cell differentiation. Our findings suggest that HSPCs serve as a reservoir for an epigenetic memory of smoking. Additionally, we observed short-term cell-specific smoking-associated DNAm changes in myeloid cells. Our results demonstrate the utility of the MSA in expanding our knowledge of both transient and persistent environmental exposure-associated DNAm changes.

Early pregnancy requires a tightly regulated pro-inflammatory environment shared between the primitive placenta and decidua. While immune balance supports successful implantation and placental invasion, disruptions in immune signaling during this period can impair implantation and lead to embryo loss. In this study, we investigated the molecular mechanisms underlying immune imbalance during implantation using a trophoblast stem cell (TSC) model. TSCs were cultured in either stem cell or syncytiotrophoblast (STB) differentiation medium and treated with either lipopolysaccharides (LPS) or interferon beta (IFNB). RT-qPCR and Western blotting revealed that LPS failed to induce a pro-inflammatory cytokine response in TSCs or STBs. In contrast, IFNB triggered a strong antiviral response in both TSCs and STBs. RNA-sequencing of IFNB-treated TSC and STB 3D spheroids revealed subtle differences between the TSCs and STB responses to interferons. Both TSC and STB IFNB-treated spheroids mount an interferon-mediated antiviral response; however, STB spheroid genes associated with the type I interferon response, viral RNA/DNA sensing, and antigen processing were upregulated. We also compared the interferon response between the CT27 (female) and CT29 (male) TSCs and STBs. While STBs showed minimal differences, the CT29 TSCs exhibited a markedly stronger interferon response than the CT27 TSCs. Collectively, these findings suggest that the primitive placenta is selectively responsive to interferon signaling rather than direct pathogen-associated stimuli. This implies that maternal immune activation, rather than microbial invasion, likely drives that placental immune response and embryo success at this stage. Understanding these dynamics underscores the importance of the maternal immune balance in early pregnancy success.

Background: Maternal micronutrient deficiencies (MNDs) and inflammation contribute to adverse birth outcomes While the individual effects of MNDs have been studied, the consequence of co-occurring MNDs remains unclear. Objectives: To examine the associations between maternal micronutrient deficiencies and inflammation with adverse birth outcomes (ABOs). Methods: Data from 5,408 pregnant women across 11 datasets from 10 countries were analyzed. Descriptive analyses explored the distribution of MNDs (iron, vitamin A, zinc, serum folate, vitamin D, and vitamin B12) and inflammation (c-reactive protein >5 mg/L or -(1)-acid glycoprotein > 1g/L) by maternal characteristics (age, height, education, socioeconomic status [SES]) using chi-square tests. Associations of 1) single MNDs and inflammation and 2) co-occurring MNDs (2 deficiencies at a time) with low birth weight (LBW, < 2500 g), preterm birth (PTB, < 37 wks), and small-for-gestational age (SGA, < 10th percentile for gestational age), were examined using modified Poisson regression to estimate relative risk (RR), adjusting for age, SES, and dataset. Results: Young maternal age and short height were associated with up to 9.7% and 25% higher prevalence of MNDs and inflammation, respectively. Lower education and SES level were associated with higher prevalence of Vitamin B12 deficiency. Women with folate deficiency had an increased risk of LBW (RR [95% CI]: 1.22 [1.06, 1.39]). Co-occurring MNDs for folate and vitamin B12 were also associated with increased LBW risk (1.38 [1,1.9]) as was folate deficiency without iron (1.28 [1.09, 1.51]) or vitamin B12 deficiency (1.67 [1.09, 2.56]) compared with mothers without either deficiency. Iron deficiency without vitamin B12 deficiency was associated with a reduced LBW risk (0.4 [0.2, 0.79]). Conclusion: Maternal MNDs, especially folate and vitamin B12, are linked to adverse birth outcomes. Complex nutrient interactions highlight the need to explore these relationships to improve maternal and neonatal health interventions.

BackgroundTryptophan (Trp) metabolism is a central immunometabolic axis in inflammatory bowel disease (IBD) and has been linked to inflammatory activity and immune regulation. While individual Trp metabolites have been associated with disease severity and treatment response, systems-level frameworks to define metabolic subtypes in IBD are lacking. ObjectiveTo identify reproducible Trp-related metabolic subtypes ("metabotypes") in IBD and assess their association with disease activity, clinical outcomes, and early disease development. DesignWe applied unsupervised clustering to serum concentrations of 16 Trp-related metabolites in a discovery cohort of patients with IBD undergoing biologic induction therapy (n=134). Metabotypes were validated in three independent IBD cohorts (total n>2,800), a healthy reference population, and a prospective cohort of first-degree relatives at risk for Crohns disease. Associations with disease activity, longitudinal outcomes, and metabolic pathways were assessed using multivariable regression and survival analysis. ResultsFour reproducible metabotypes with distinct metabolite profiles were identified across cohorts: Low Kyna, High Kyna, High Quin, and Balanced. Low Kyna and High Quin metabotypes were consistently associated with increased inflammatory activity and adverse clinical outcomes, including increased risk of treatment escalation and disease progression. Pathway-level analyses revealed alterations in NAD-related, lipid, and amino acid pathways between inflammatory metabotypes. A metabotype resembling inflammatory disease states was enriched in individuals who later developed Crohns disease in a prospective pre-disease cohort. ConclusionTrp-linked metabotypes define reproducible immunometabolic states in IBD that associate with disease activity and clinical outcomes and may precede disease onset. These findings provide a framework for metabolic stratification and biomarker-guided clinical trials targeting immunometabolic pathways. What is already known on this topicTryptophan metabolism through the kynurenine pathway is a central immunometabolic axis in inflammatory bowel disease (IBD) and has been linked to inflammatory activity and immune regulation. Individual tryptophan metabolites have been associated with disease severity and treatment response, but their clinical utility for patient stratification remains limited. Systems-level approaches to define clinically meaningful metabolic subtypes in IBD are lacking. What this study addsWe identify four reproducible tryptophan-related metabolic subtypes ("metabotypes") that are consistently associated with disease activity across multiple independent IBD cohorts. Inflammation-associated metabotypes show distinct pathway-level alterations, including differences in NAD-related metabolism and broader metabolic programs. A metabotype resembling inflammatory disease states is detectable before clinical diagnosis in individuals who later develop Crohns disease. How this study might affect research, practice or policyMetabotype-based classification provides a framework for molecular stratification of patients in mechanistic studies and clinical trials targeting immunometabolic pathways. This approach may support biomarker-guided monitoring of disease activity and disease progression in IBD. Identification of preclinical metabolic states highlights the potential of metabolomics for early disease detection and prevention-oriented research strategies.