Mitochondrion

Background: Shotgun metagenomic pipelines routinely discard host-derived reads, which can exceed 90% of reads in low-biomass clinical samples. Whether these reads support mitochondrial heteroplasmy detection, and at what allele fraction, has not been established. Methods: Shotgun metagenomic data from 47 upper respiratory tract swabs from healthy adults in Nagpur, India, were analysed. Human Reads were recruited against the rCRS and realigned competitively to GRCh38 to remove nuclear mitochondrial segments (NUMTs). Kraken2 was used as a microbial control. Heteroplasmy was called against a pooled, substitution-specific beta-binomial error model and a leave-one-out panel of normals. The detection limit was evaluated from the substitution spectrum against gnomAD v3.1. Results: Complete mitochondrial genomes were recovered from all samples (mean depth 433X, range 9-3,029X). Competitive realignment relocated 3.22% of recruited reads to nuclear loci; microbial cross-mapping removed 0.045%. Error rates varied 12.8-fold across substitution classes, with T>A being the highest. The 134 calls had a Ti/Tv of 0.43, compared with 3.51 in gnomAD, and T>A accounted for 42.5% of calls. T>A allele fractions never exceeded 4.72%, whereas transitions reached 90.81%. At a 5% threshold, T>A calls disappeared, and Ti/Tv rose to 2.20 before plateauing; 16 calls exceeded this limit. Macrohaplogroups were assigned in 46 of 47 samples (M 22, R 23, N 1). Conclusions: Metagenomic host reads support complete mitochondrial recovery, haplogroup typing, and heteroplasmy detection for allele fractions > 5%. Below this, a T>A-dominated error process persists after NUMT removal that background-fitted models do not capture.

Background. Alternative oxidase (AOX) is a mitochondrial terminal oxidase that provides an alternative route for electron transfer, contributing to respiratory flexibility and redox homeostasis under stress conditions. In trypanosomatids, AOX displays a highly uneven distribution associated with extensive diversification of mitochondrial electron transport chains (ETCs). In addition to canonical AOX, many trypanosomatids encode an enigmatic AOX-like protein (AOX-L), whose evolutionary origin and functional significance remain unresolved. Results. Here, we investigated the evolutionary distribution, phylogenetic relationships, structural conservation, and functional divergence of AOX and AOX-L across euglenozoans. Comparative genomic analyses revealed a patchy distribution of both proteins among kinetoplastids, with multiple independent losses during evolution. Phylogenetic analyses demonstrated that AOX and AOX-L represent distinct evolutionary lineages, indicating independent origins rather than duplication-derived divergence. While AOX showed a conserved eukaryotic origin, AOX-L occupied a separate phylogenetic position and lacked key residues required for ubiquinol oxidation. Structural modelling revealed that AOX-L retained the characteristic AOX fold, predicted membrane association, and dimeric organization, but lacked the catalytic architecture necessary for enzymatic activity. Transcriptomic and biochemical analyses of four AOX-containing trypanosomatid species showed that AOX expression and activity correlated with mitochondrial ETC organization. Species lacking cytochrome-dependent complexes III and IV displayed substantially higher AOX expression and enzymatic activity, consistent with AOX functioning as the primary terminal oxidase in these lineages. Conversely, species retaining a canonical ETC exhibited lower AOX activity, suggesting a role in metabolic flexibility and redox regulation. Conclusions. Our study provides a comprehensive evolutionary and functional framework for alternative oxidases in trypanosomatids. We demonstrate that AOX and AOX-L are evolutionarily distinct proteins with different predicted functions: AOX maintains respiratory electron flow according to lineage-specific mitochondrial requirements, whereas AOX-L represents a structurally conserved but catalytically inactive protein family that may have acquired an alternative regulatory role. These findings highlight how mitochondrial respiratory components diversify during eukaryotic evolution and provide a basis for future investigations into the biological function of AOX-L.

Mitochondrial genomes of angiosperms are unusual due to their large size, low gene content, and high structural variation. In the palm family (Arecaceae), mitochondrial architecture is highly dynamic, characterized by expansive sizes, fragmentation, and extensive recombination activity. This biological complexity presents a significant bioinformatic challenge, especially since only three complete palm mitochondrial genomes are publicly available to date. Here, we present the complete mitochondrial genome sequence for the Amazonian palm Euterpe precatoria, assembled de novo using PacBio HiFi long-read sequencing data. The assembly required extensive manual curation and graph tracing to resolve a complex recombination topology. The final resolved sequence spans 1,268,308 base pairs and is supported by a mean read coverage depth estimated at 746X. Annotation revealed a high degree of structural complexity, including 177 total gene features corresponding to 76 unique genes. This gene set includes 41 unique protein-coding genes (56 features), 32 unique tRNA genes (118 features), and three unique rRNA genes, reflecting the characteristic gene duplication and fragmentation documented in Arecaceae. The complete mitochondrial genome sequence is publicly available and provides a high-quality resource for understanding the mechanisms of plant mitochondrial genome expansion and structural evolution.

Methylmalonic aciduria (MMA) is caused by deficiency of methylmalonyl-CoA mutase (MMUT), which catalyses the final step of propionate catabolism and fuels the tricarboxylic acid cycle for energy production. Previous studies reported disrupted mitochondrial homeostasis in MMA, including reduced mitochondrial membrane potential and increased oxidative stress, especially in energetically demanding and chronically affected tissues like brain and kidneys. However, how these changes impact mitochondrial energy production remains unclear. Here, we systematically investigated mitochondrial energy production using extracellular flux analysis in cellular models of MMA, including 293T cells, patient-derived fibroblasts, urine-derived epithelial kidney cells and induced pluripotent stem cells (iPSCs) as well as iPSC-derived neurons harbouring either complete loss (knockout, KO) or pathogenic missense variants in MMUT. We found no impact on mitochondrial energy production in MMUT-KO 293T cells and fibroblasts compared to controls. In contrast, fibroblasts and 293T cells expressing the pathogenic MMUT-p.N219Y variant showed decreased energy production. This corresponds to the altered mitochondrial membrane potential in 293T cells, but contrasts with their unchanged mitochondrial abundance. Depletion of glucose, glutamine and pyruvate from the media or provision of each individually as sole fuel source exacerbated the phenotype of MMUT-p.N219Y clones, but did not induce a phenotype in MMUT-KO 293T cells. Finally, we confirmed reduced mitochondrial energy production in patient-derived kidney cells but found no evidence in MMUT-p.N219Y iPSCs and their derived neurons. Overall, our work suggests that the impact of MMUT-deficiency on mitochondrial energy production is cell type- and variant-dependent. Further investigations should clarify molecular mechanisms and their clinical impact.

Background: Protein-coding DNA exhibits characteristic three-base periodicity. In unaligned DNA libraries, however, this signal should cancel if fragment boundaries are uniformly distributed across codon phases. Yet, we find that in multiple metagenomic libraries, base frequencies depended systematically on distance from fragment boundaries, producing a period-3 pattern. We investigated the origin of this unexpected signal. Results: The signal was observed in modern metagenomic datasets and widely across ancient-DNA datasets. We developed a new alignment-free algorithm to infer latent triplet-phase structure from nucleotide composition. The inferred shifts were strongly nonuniform at fragment boundaries, explaining the read-coordinate periodicity. We initially suspected a protocol artifact. To test an alternative, we derived a probabilistic framework linking nucleotide-dependent boundary selection to codon-phase frequencies. The framework showed that, because nucleotide composition differs among codon positions, preferential breakage at particular nucleotides biases the phases represented at fragment boundaries. Simulations confirmed this mechanism: purine-associated fragmentation produced strong period-3 coherence in a coding-dense bacterial genome, whereas no comparable genome-wide effect appeared in the human reference genome. Conclusions: The observed read-coordinate three-base periodicity does not require an intrinsic phase bias in the source DNA. Instead, sequence-dependent fragment formation or recovery can couple boundaries to codon phase. Purine-associated post-mortem fragmentation plausibly explains the widespread phenomenon in ancient-DNA libraries, although PCR- or library-specific mechanisms may contribute to it in modern datasets. Our alignment-free algorithm provides a practical method for estimating and normalizing phase shifts before searching for positional nucleotide patterns, preventing boundary-associated bias from being mistaken for an intrinsic biological pattern.

Mitochondrial disease arises from variants in either the mitochondrial or nuclear genome that impair mitochondrial oxidative phosphorylation. Complex III (ubiquinol:cytochrome c oxidoreductase, CIII2) is a key enzyme of the mitochondrial respiratory chain, catalyzing electron transfer from reduced coenzyme Q10 (ubiquinol) to complex IV, via cytochrome c. CIII2 has one subunit encoded by mitochondrial DNA, the remainder being nuclear encoded. Individuals with CIII2 deficiency present with significant clinical heterogeneity, on account of pathogenic variants in genes encoding structural CIII2 subunits or assembly factors. UQCC1 (GenBank: NM_018244.5) encodes the mitochondrial complex III assembly factor Ubiquinol-cytochrome c reductase complex assembly factor 1, which is currently not associated with human disease. Here we describe nine subjects from eight unrelated families harboring biallelic UQCC1 variants with heterogeneous, early-onset mitochondrial disease presentations and lactic acidosis. Subject-derived fibroblasts demonstrated a decrease in the steady-state levels of UQCC1 and its interacting partner UQCC2, with structural modelling supportive of variant pathogenicity. BN-PAGE and quantitative proteomics demonstrated decreased CIII2 abundance whilst mass spectrometry (MS)-complexome profiling corroborated these findings, identifying impaired respiratory supercomplex formation and a delay in the incorporation of newly synthesized CIII2 subunits into native assemblies. Lentiviral transduction experiments in subject-derived fibroblasts restored the levels of both UQCC1 protein and CIII2 activity. Collectively, these findings provide convergent genetic, biochemical, proteomic and functional evidence that bi-allelic UQCC1 variants disrupt CIII2 assembly, establishing UQCC1 deficiency as a cause of mitochondrial CIII2 deficiency.

Changes of DNA sequence in the human genome may contribute to human aging, but it has been difficult for the field to define the details of the contribution. Cancer cells have altered ratios of DNA damage and repair pathways, and thus it is inappropriate to utilize them as models for normal cells. Also, somatic mutations from each lineage cannot be clearly identified in cell populations with multiple lineages. Normal human colon crypts are one of the few mini-organ systems where monoclonal cell populations can be found for somatic mutation analysis. In a parallel study, we described complex insertion/deletion (indel) events in normal human colon. Here we focus on simple deletions that lack any nucleotide addition or adjacent base substitutions. We find that ~58% of the simple deletions are consistent with a strand slippage event, and the remainder provide insights into other important repair processes. Among the 4,264 >=2-nucleotide simple deletions, ~71% are most consistent with nonhomologous DNA end joining (NHEJ), with <1.5% of the events being more consistent with alternative end joining (aEJ). The incidence of NHEJ-type simple deletions is ~8-fold more frequent than the complex indels that also show NHEJ features. Similar to the complex indels, the simple deletions show a linear trend of event accumulation (positive slope) with an overall ~10-fold increase in absolute number across the human lifespan, 0.8 months to 90 years old, for individuals with no radiation or chemotherapy treatment. Interestingly, radiation treatment increases non-slippage deletions with little effect on slip deletions.

Background/Objectives: Aldehyde dehydrogenase 2 (ALDH2), a mitochondrial enzyme, mitigates cellular stress by detoxifying reactive aldehydes produced during alcohol exposure and endogenous metabolic processes. ALDH2*2 polymorphism, which results in deficient catalytic activity, is prevalent in East Asian populations and is associated with an increased risk of cancer and other diseases. While the effects of ALDH2 deficiency on cellular stress responses are well documented, the specific metabolic dysregulations arising from this deficiency require further investigation. Methods; An ALDH2 knockout subline of SKBR-3 cells was established using CRISPR/Cas9 strategy. The metabolomic profiles of control (SKBR-3/C) and ALDH2 knockout (SKBR-3/ADKO) cells were evaluated via ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS). Results: ALDH2 knockout in SKBR-3 cells resulted in increased cellular stress, evidenced by elevated levels of 8-OHdG, reactive oxygen species (ROS), and malondialdehyde (MDA). Metabolomic analysis of the paired cell lines assigned a total of 5,033 signals, of which 281 were significantly different (fold change [&ge;] 2, p-value <0.05) between the two groups. Pathway analysis with MetaboAnalyst identified significant metabolic pathways associated with ALDH2 deficiency, particularly those involving arginine biosynthesis, and arginine and proline metabolism. ALDH2 deficiency reprogramed arginine metabolism toward polyamine pathways while suppressing the urea cycle. Network analysis further revealed alterations in amino acid and lipid metabolism in ALDH2-deficient cells. Conclusions; This study provides a novel insight into the role of ALDH2 in cancer metabolism and may have broader implication for cancer biology.

Despite recent evidence of the ability of systemic and organ-specific proteomic aging clocks to predict a large range of age-related diseases, their utility in cancer research remains largely underexplored. In this study, we evaluated the predictive ability of systemic and organ-specific proteomic aging clocks for (i) all-cause mortality, (ii) cancer-related mortality, and (iii) cancer incidence in a sample of Finnish twins (N = 401, mean age at baseline 62.3 years) with Olink proteomic data. Participants were followed for a mean of 10.6 years (SD +- 1.6), with estimates from four systemic and 18 organ-specific proteomic aging clocks calculated at baseline. Cox proportional hazards regression models adjusted for sex and age at baseline identified 15 proteomic aging clocks as significant predictors of all-cause mortality (Ncases = 28) after multiple testing correction (FDR < 0.05), with 11 remaining significant after lifestyle adjustment (Ncases = 27). For several clocks, effect sizes were large, with hazard ratios (HR) approximately doubling per one standard deviation (SD) increase in clock estimates. In predicting cancer-related deaths among participants without a prior cancer diagnosis at baseline (Ncases = 9), only the Immune clock showed a significant positive association after multiple testing correction in basic (HR = 2.94, 95% CI: 1.88-4.59) and lifestyle-adjusted models (HR = 3.31, 95% CI: 1.78-6.17). No associations with cancer incidence (Ncases = 34 basic, Ncases = 31 adjusted) survived multiple testing correction, although several nominal associations were observed. In conclusion, our findings suggest that proteomic aging clocks are strong predictors of all-cause and cancer-specific mortality, independent of lifestyle factors. Proteomic aging clocks may further serve as valuable tools for stratifying cancers by mortality risk.

Myotonic dystrophy type 1 (DM1) is caused by a CTG expansion in the 3' untranslated region of the DMPK gene that results in the expression of expanded CUG RNA which trap splicing factors resulting in widespread alternative splicing dysregulation. A panel of these splicing events, or splicing index (SI), is currently used to measure splicing dysregulation in DM1 patients. Despite significant interest in splicing, little is known about its progression over time in DM1. The objective of this study was to evaluate the changes in SI over 3 years in a well-phenotyped cohort of DM1 participants and identify factors contributing to these changes. RNA was extracted from vastus lateralis (VL) biopsies of 20 DM1 participants taken 3 years apart and the SI score was measured using targeted RNA sequencing. Baseline SI scores correlated with percent predicted muscle strength of the ankle dorsiflexors, knee extensors and hand grip. On average, SI scores worsened over 3 years, but the progression varied markedly among participants. Adult/juvenile phenotypes and lower baseline SI scores were among the factors associated with higher progression of SI scores over time. These findings emphasize the importance of integrating clinical phenotype and baseline molecular severity when interpreting longitudinal SI change, particularly in the context of clinical trials.

BackgroundClonal hematopoiesis of indeterminate potential (CHIP) is associated with ageing and atherosclerotic coronary artery disease (CAD), but evidence has largely come from peripheral blood. Whether somatic mutational profiles differ between arterial tissue and blood, and whether hematopoietic clones are represented in arterial tissue, remain poorly understood. MethodsWe performed deep whole-exome sequencing on 302 blood granulocytes and 263 arterial tissues from patients undergoing coronary artery bypass graft surgery, including 221 matched blood-tissue pairs, with mean sequencing depths of 130.5X and 120.8X, respectively. Somatic variants were identified using Mutect2. Associations with age and CAD complexity, assessed using SYNTAX score, were evaluated using multivariable regression. Primary analyses focused on variants with variant allele fractions of 10-25%. ResultsCHIP was detected in 24/302 (7.9%) blood granulocytes, predominantly involving DNMT3A, TET2, and ASXL1. In contrast, somatic mutations in CHIP-associated genes in arterial tissues showed a distinct profile dominated by CALR, SETDB1, and PDSS2. Somatic mutation carrier status increased with age in blood ({beta} = 0.07, p = 0.02) and arterial tissues ({beta} = 0.06, p = 0.04). Blood CHIP was associated with lower SYNTAX score ({beta} = -1.05, p = 2.66 x 10-3), partly driven by DNMT3A carriers ({beta} = -1.06, p = 0.04), whereas arterial CALR mutations were associated with higher SYNTAX score ({beta} = 1.14, p = 0.03). Among 221 matched pairs, only four identical variants, involving CHEK2, DNMT3A, GNB1, and SRSF1, were detected in both compartments. Genome-wide analyses showed compartment specificity, with bile acid metabolism enriched among arterial tissue-specific mutations (PAdj = 0.038). ConclusionBlood and arterial tissues in CAD harbour distinct somatic mutational landscapes with limited cross-compartment sharing. These findings suggest that cardiovascular somatic mutations may reflect both hematopoietic and arterial tissue-associated processes and motivate cell-resolved and spatial studies to define their origins and roles in cardiovascular ageing.

Background: Barth syndrome (BTHS) is an X-linked mitochondrial disorder caused by loss-of-function mutations in TAFAZZIN (TAZ), resulting in defective cardiolipin (CL) remodeling, cardiomyopathy, and premature death. The mechanisms linking TAZ deficiency to impaired mitochondrial quality control remain incompletely understood, and there are no disease-modifying therapies available for BTHS. Here, we investigated the role of Acyl-CoA Lysocardiolipin Acyltransferase-1 (ALCAT1), a stress-inducible phospholipid-remodeling enzyme, in BTHS cardiomyopathy. Methods: Inducible TAZ knockdown (TAZKD) and cardiomyocyte-specific TAZ knockout (TAZcKO) mice were used to determine the effects of genetic ALCAT1 deletion or pharmacological inhibition with Juvenatin, a highly selective small molecule ALCAT1 inhibitor. Cardiac function, exercise capacity, mitochondrial function, lipid remodeling, lysosomal function, and mitophagic flux were assessed. Results: ALCAT1 was markedly upregulated in TAZ-deficient hearts. Genetic ALCAT1 deletion attenuated cardiac dysfunction in TAZKD mice. Juvenatin similarly improved cardiac function in TAZKD mice and reversed established cardiomyopathy in TAZcKO mice. Genetic or pharmacological ALCAT1 inhibition improved mitochondrial ultrastructure and respiration and reduced mitochondrial oxidative stress. These benefits occurred without correcting the characteristic CL abnormalities caused by TAZ deficiency. Instead, ALCAT1 deletion or inhibition corrected pathological phosphatidylinositol remodeling, normalized phosphoinositide signaling, and restored lysosomal function and mitophagic flux, leading to improved mitochondrial quality control in TAZ-deficient cells. Conclusions: ALCAT1 contributes to BTHS cardiomyopathy through a CL-independent pathway involving dysregulated phosphatidylinositol/phosphoinositide signaling, lysosomal dysfunction, and impaired mitophagy. Genetic and pharmacological targeting of ALCAT1 restores mitochondrial and cardiac function despite persistent CL abnormalities, establishing ALCAT1 as a promising disease-modifying therapeutic target for BTHS.

Transfer RNA (tRNA) modifications regulate protein synthesis, yet their sequence positions and stoichiometries are known only for a subset of tRNAs in model organisms. Aplysia californica is a key neurobiological model in which neuronal modified ribonucleosides have been linked to changes in animal behavior. However, Aplysia tRNA modifications have not been mapped, obscuring their roles in modulating neuronal functions. To address this knowledge gap, we leveraged hybridization pulldowns and an integrated mass spectrometry (MS) strategy combining nucleoside profiling, intact tRNA measurements, and bottom-up tRNA modification mapping to determine the full sequences of Aplysia tRNAGluCUC and tRNALysUUU. Sub-stoichiometric modifications including dihydrouridine (D), 5-methylcytidine (m5C), 5,2'-O-dimethyluridine (m5Um) and 1-methyladenosine (m1A) on tRNAGluCUC varied across tissues, suggesting tRNA modifications are regulated to accommodate translational needs. In addition to conserved thio-modifications in the anticodon loop of tRNALysUUU, we report 2-thiocytidine (s2C) at position 32, representing the first detection of s2C in eukaryotes. We also developed a generalizable method to directly detect tRNA halves by intact LC-MS, revealing endogenous tRNA cleavage patterns. Our findings illustrate the advantages of combining intact and bottom-up MS, reveal s2C as an unexpected tRNA modification in a classical neurobiological model and offer new insights into post-transcriptional regulation of animal physiology.

Age-related decline in skeletal muscle function, often manifesting as slower gait speed, contributes significantly to frailty and loss of independence. To uncover the molecular basis of this decline, we investigated coordinated changes across the transcriptome, miRNome, and methylome in vastus lateralis muscle from female vervet monkeys (Chlorocebus aethiops sabaeus), contrasting middle-aged animals with normal gait speed versus older animals with declining gait speed. Through multi-omics integration, we identified a molecular signature that sharply distinguishes these two groups. This signature involves key regulatory molecules, including miR-181a-5p, miR-320b and miR-425 control of SYNCRIP, miR-193b-3p regulation of MCL1 and PLAU, as well as a multi-omic regulatory network governing ZNF274 and ANP32E gene expression, and altered levels of mRNAs such as VEZT. Functional annotation implicates miRNA signatures with inverse expression of their mRNA targets operating within systems of skeletal muscle biology, metabolism, and potential immune-inflammation mechanisms. Collectively, these findings reveal an interconnected network of epigenetic, post-transcriptional, and transcriptional alterations associated with impaired muscle function during aging. This molecular signature provides novel insights into potential mechanisms driving sarcopenia and highlights specific molecules as potential targets for interventions aimed at preserving muscle health.

Photosynthetic electron transport is tightly regulated to balance ATP and NADPH production and prevent over-reduction of the photosynthetic machinery. How this balance is maintained under severe ATP limitation, particularly under anoxia, is not fully resolved. Here, we used several deletion mutants of chloroplast ATP synthase subunits from Chlamydomonas reinhardtii to examine how electron flow is reorganized under anoxic conditions through the interplay between linear electron flow, cyclic electron flow, and alternative electron sinks. Using a combination of optical spectroscopy methods and membrane inlet mass spectrometry, we characterized changes in photosynthetic electron transport, proton motive force relaxation and gas exchange. Under oxic conditions, the {Delta}atpH mutant (lacking the c-subunit) showed strong acceptor-side limitation at photosystem I (PSI), whereas atpC1-2 (lacking the {gamma}-subunit) resembled the wild type. Under anoxia, this limitation was partially relieved, coinciding with sustained electron transport and prolonged hydrogen production. In particular, the {Delta}atpH mutant exhibited increased cyclic electron flow under oxic conditions and maintained PSI activity under anoxia despite impaired ATP synthesis. Prolonged hydrogen production was also observed in atpC1-2, which did not share the increased respiration of {Delta}atpH. Hydrogenase activity under anoxia is consistent with a role as an electron sink that may help alleviate PSI acceptor-side limitation and rebalance the redox state of the electron transport chain. These findings show that ATP synthase deficiency reorganizes photosynthetic electron flow and promotes prolonged hydrogen production, and point to hydrogenase as a candidate electron sink under energy-constrained conditions.

Recent lifespan frameworks of aging theorize that pubertal development and cellular aging are governed by overlapping pathways, but the time lag between adolescence and older adulthood complicates the study of shared molecular mechanisms. Here we compare a DNA-methylation-based epigenetic index of pubertal development to epigenetic clocks, which were designed to measure biological aging, as a putative biomarker of adult aging and health in two adult cohorts: 3,642 older Americans in the Health and Retirement Study (HRS; age range, 56 to 100 years; 2,106 females) and 18,676 adults from Generation Scotland (GS; age range, 18 to 98 years; 10,974 females). Epigenetic puberty was correlated with chronological age in both cohorts, moderately in the older HRS sample (r = 0.51 in females, r = 0.51 in males) and strongly across the wider adult age range of GS (r = 0.82 in females, r = 0.86 in males). In both cohorts, age-residualized epigenetic puberty was associated with accelerated biological aging as measured by epigenetic clocks and other aging biomarkers (standardized coefficients: HRS, 0.13 to 0.47, all P < 0.005; GS, 0.05 to 0.40, all P < 0.001). Epigenetic puberty was linked with multiple health outcomes: mortality in HRS in both sexes (ORs, 1.39 and 1.46, both P [&le;] 0.001) and in GS among females (HR, 1.12, P = 0.003); 10-year onset of diabetes, any cancer and lung disease in GS (HRs, 1.12 to 1.19, all P < 0.005); and with multimorbidity, functional limitations and BMI in females in HRS (standardized coefficients, 0.10 to 0.16, all P < 0.001). It also accounted for a unique, albeit smaller, proportion of variance in aging-related outcomes beyond that explained by epigenetic clocks. These findings indicate that the epigenetic puberty index reflects pro-aging underlying biology, showing overlapping molecular pathways between pubertal development and cellular aging, and support a lifespan-based approach to studying biological aging.

Lipid droplets (LDs) play a central role in cellular carbon and energy homeostasis, in particular through the roles played by numerous LD-associated proteins. A long-standing question concerns how these proteins are molecularly organized at the seemingly overcrowded LD surface. Delayed in TAG Hydrolysis 1 (DTH1) has been identified as a key protein for efficient LD degradation in Chlamydomonas reinhardtii, however the underlying molecular mechanism remains unknown. DTH1 contains several structural features including a coiled-coil domain, a phosphatidylethanolamine (PE) specific lipid-binding domain, intrinsically disordered regions, and an alpha-helical C-terminal domain, that make it a compelling candidate for acting as a molecular scaffold in protein recruitment and/or organelle tethering. In this study, we used the Chlamydomonas knock-out mutant dth1 as an entry point to begin dissecting the molecular organization of proteins at the LD surface. Comparative lipidomic and proteomic analyses of isolated LDs from both WT and dth1 mutants revealed several key features associated with LD degradation during nutrient recovery. First, PE emerged as an important lipid species involved in LD degradation. Second, active communication occurs among multiple subcellular organelles particularly between LDs, peroxisomes and mitochondria. Third, DTH1 was found to play a critical role in remodeling both the LD proteome and lipidome. Furthermore, detailed analyses of LD degradation during nitrogen recovery uncovered the formation of micro-LDs, a phenomenon not yet reported in microalgae. Collectively, these results highlight the tight coordination of metabolic activities across subcellular compartments and emphasize the importance of intracellular carbon trafficking during stress recovery.

Purpose. Neuroblastoma is the most common malignancy of infancy, and cancer cells strongly depend on iron metabolism for proliferation. Whether sub-cytotoxic iron load reprograms mitochondrial and redox metabolism, and how this relates to the malignant behavior of neuroblastoma cells, is poorly defined. We hypothesized that exposure of human neuroblastoma cells to a non-toxic iron overload perturbs mitochondrial and redox metabolism, thereby affecting cancer cell properties. Methods. SH-SY5Y neuroblastoma cells were exposed to 100 M iron sulfate for 24 h. The resulting changes were monitored using complementary methods, including cell viability, cytotoxicity, proliferation and migration assays, intracellular iron quantification by ICP-MS, confocal imaging of mitochondrial morphology and membrane potential ({Delta}{Psi}m), high-resolution respirometry with flux control ratios, probe-based oxidant and H2O2 measurements, and label-free quantitative proteomics with Ingenuity Pathway Analysis. Results. Non-toxic iron loading raised intracellular iron about four-fold, promoted cell proliferation and reduced migration. Iron induced mitochondrial fragmentation and {Delta}{Psi}m depolarization while preserving mitochondrial content and OXPHOS coupling efficiency. Among respiratory states, only proton-leak respiration increased, paralleled by a higher leak/ETS coupling ratio. Total cellular oxidant levels and H2O2 output were strongly reduced. The proteome showed downregulation of ribosomal proteins and translation/RNA-processing components and upregulation of histones and ferritin. Conclusion. Mild iron exposure reprograms mitochondrial and redox metabolism in neuroblastoma cells. The observed selective increase in proton leak lowers {Delta}{Psi}m to a range that biophysically limits oxidant production while OXPHOS coupling is preserved. Interfering with this adaptive, proton leak-dependent and preventive antioxidant program may offer a way to alter neuroblastoma progression.

This systematic review aimed to synthesize evidence on the association between DNA methylation (DNAm) age and all-cause and disease-specific mortality risks in adults. A systematic search of six databases from Jan 1 2011 to April 15 2026 identified articles assessing the association of DNAm age measures and all-cause or disease-specific mortality in adults from general or disease-specific populations. Effect sizes were pooled as hazard ratios (HRs) with 95% confidence intervals (CIs) using inverse-variance random-effects models. Of 42 023 records screened, 125 articles (380 705 participants) met the inclusion criteria, with 53 included in meta-analyses. Overall, 48 distinct DNAm clocks were evaluated. Higher DNAm ages were associated with higher risks of all-cause, cardiovascular, and cancer mortality risks. In general populations, GrimAge epigenetic age acceleration (EAA) showed consistent associations across mortality outcomes, with each 1 year higher GrimAge EAA associated with ~6-10% higher all-cause (31 cohorts, n=53 642, HR per SD: 1.46 [95% CI 1.38, 1.55], HR per 1-year: 1.09 [95% CI 1.08, 1.11], I^2=84.1%, p-value=1.21 x 10^(-14)), cardiovascular (6 cohorts, n=30 751, HR per SD: 1.55 [95% CI 1.38, 1.73], HR per 1-year: 1.10 [95% CI 1.07, 1.13], I^2=28.0%, p-value=1.71 x 10^(-4)), and cancer (5 cohorts, n=27 187, HR per SD: 1.31 [95% CI 1.15, 1.50], HR per 1-year: 1.06 [95% CI 1.03, 1.10], I^2=33.6%, p-value=4.76 x 10^(-3)) mortality. Clocks incorporating additional surrogates and technical refinements showed numerically larger pooled associations than their original counterparts. GrimAge2 (HR per SD: 1.65 [95% CI 1.53, 1.77] vs 1.46 [1.38, 1.55]) and PCGrimAge (1.56 [1.36, 1.78] vs 1.51 [1.32, 1.74]) showed higher pooled HR association with all-cause mortality than GrimAge. Further work is warranted to evaluate the responsiveness of DNAm clocks to interventions and potential clinical utility.

Translation initiation is a critical stage of protein synthesis in prokaryotes, where ribosomes assemble on mRNA to begin decoding genetic information. The interactions between ribosomal RNA (rRNA) and mRNA significantly influence this process, with features like ribosomal binding site (RBS) affecting ribosomal dynamics. However, direct experimental validation of these types of interaction is yet to be provided. In this study, we developed an optimized small sub-unit sequencing (SSU-seq) protocol for Escherichia coli, enabling the detailed examination of ribosomal complexes and subunits dynamics. Unlike Ribo-seq, which primarily maps fully assembled ribosomes, SSU-seq captures both ribosomal subunits and intact ribosomes, offering a comprehensive view of translation initiation and elongation events. By integrating experimental data with predictive models, our study advances the understanding of translation regulation in prokaryotes and underscores the utility of SSU-seq in decoding the complexities of ribosome-mRNA interactions. Our analysis reveals distinct patterns of small subunit distribution across the entire mRNAs with higher density near positions with stronger rRNA-mRNA hybridization. The results may suggest that rRNA-mRNA hybridization positions along the entire transcript facilitate efficient start codon recognition by maintaining proximity between the small subunit and the mRNA.