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Alzheimer's disease (AD) arises from heterogeneous biological processes, and long-term environmental exposures may become biologically embedded, as reflected in epigenetic clocks. We calculated blood DNA methylation-based epigenetic clocks and constructed protein co-abundance networks of cerebrospinal fluid (CSF) proteomics data. We performed association analysis of epigenetic age acceleration with network modules, followed by functional and cell-type enrichment analyses, association analyses of hub proteins with AD endophenotypes, and pseudotime trajectory analysis. Six network modules were significantly associated with epigenetic age acceleration and were enriched in pathways related to neuronal connectivity, proteostasis, immune activation and remodeling, immune signaling, and immunoepigenetic regulation. Hub proteins demonstrated significant associations with baseline amyloid/tau/neurodegeneration biomarkers and longitudinal cognitive changes. Pseudotime analysis revealed continuous, non-linear variation in epigenetic age acceleration along the inferred trajectory. Our CSF proteomics study identified neuronal, proteostatic, and immune-related molecular signatures associated with epigenetic age acceleration in AD.

Uveal melanoma (UM) is a challenging malignancy, in terms of diagnosis, risk stratification, and treatment associated with high morbidity and mortality rates. It has been demonstrated that E2F-related pathways play a significant role in the tumorigenesis and distant metastasis of UM. In this study, the E2F target-related genes were utilized to construct and validate a prognostic risk score for patients with UM. Using the TCGA-UVM cohort (n = 80), 192 E2F target genes were screened using gene set enrichment analysis (GSEA) to identify survival-associated genes. Prognostic genes were filtered using Kaplan-Meier analysis, univariate Cox regression, and LASSO, followed by multivariate Cox regression to construct a risk score model. The model was validated using the GSE22138 cohort (n = 63). Functional annotations of the risk score and its impact on stratifying tumor immune microenvironment components were assessed. A total of 9 genes (CDC25B, NME1, RFC2, PRDX4, NASP, UBE2S, PRKDC, MCM6, and LBR) passed the model construction pipeline. The risk score categorization system showed an independent prognostic power (HR: 2.34, 95% CI: 1.11-4.90, p = 0.025) and a good predictability of the survival outcome (receiver operating characteristic curve analysis: area under the curve = 0.730, 95% CI: 0.60-0.86, p = 0.002). When analyzing the most frequently mutated gene cohorts, the risk score was significantly lower in the mutated subgroups of GNAQ, SF3B1, and EIF1AX. In contrast, the risk score was notably higher in the BAP1 mutated subgroup. Copy number analysis of chromosomal arms showed significant correlations between the risk score and 1q, 3q, 3p, 6p, 8q. The high-risk group showed significant infiltration for NK cells, plasma B cells, gamma delta T cells, follicular T cells, M1 and M2 macrophages with lower infiltration of common myeloid progenitor cells. In addition, the high-risk group showed higher immune and microenvironment scores. The developed E2F target-related gene model offers a robust tool for predicting the prognosis of UM patients. As a potential risk stratification method for UM, this model could have clinical applications pending further evaluation.

Many genes exhibit circadian rhythms in expression. The amplitude of oscillation, both in core clock and circadian output genes, may differ from person to person. Mutations in core clock genes are known to alter global rhythmic properties, and researchers often informally discuss "circadian amplitude." Yet, it remains unclear whether, in the general population, differences in transcriptional amplitude are largely gene-specific, or if they reflect a global, transcriptome-wide pattern-whether some individuals have globally higher or lower amplitude across the set of all rhythmic genes. We used Cosinor regression to reanalyze 4 human skin time-series transcriptomic datasets (paired epidermis/dermis samples, N = 11, N = 19) and found that, using either absolute or relative amplitude measures, distributions of gene amplitudes tended to cluster by subject. Using a non-parametric, permutation-based statistical test, we found that in many subjects this global amplitude trend was statistically significant (p ≤ 0.01). Furthermore, we found that when rhythmic genes were divided into 2 sets based on peak time (genes peaking before-noon and after-noon), the subjects' global amplitude in one set predicted global amplitude in the other set (p ≤ 0.05). We also found that in the paired epidermis/dermis datasets, subjects' global amplitude in epidermis predicted their global amplitude in the dermis (p ≤ 0.05). After identifying these trends in the skin datasets, we then found that evidence for subject-specific transcriptional rhythm strength replicated across 6 additional human time-course datasets from adipose, muscle, and blood. Perhaps surprisingly, across datasets, we found that neither established metrics of core clock transcriptional organization nor the amplitude of core clock transcription was strongly correlated with subject-specific global amplitude.

In most Bacillus thuringiensis strains, the cry genes are transcribed by RNA polymerases containing sporulation-sigma factors E or K, leading to the formation of an insecticidal crystal within the mother cell along spore development. The kurstaki HD1 strain, a parent of commercial strains, also releases the insecticidal proteins Cry1I and Vip3A in the extracellular medium. vip3A expression is activated by the transcriptional regulator VipR at the onset of the stationary phase. Here, we expanded the VipR regulon in strain HD1 by identifying the VipR-binding box upstream from the cry2Aa, cry2Ab, and cry1Ia genes, and conducting transcription assays. Unexpectedly, a VipR box was located in the promoter of a putative N-acetylmuramoyl-l-alanine amidase (ami) gene upstream from cry1Ac in strain kurstaki HD73, closely related to the HD1 but devoid of vipR. Introduction of vipR in this strain led to the expression of the ami-cry1Ac operon, resulting in an early and increased production of Cry1Ac. We demonstrated that Cry1Ac was also produced in a VipR-dependent manner in an HD73 ∆spo0A mutant. Similarly, an HD1 ∆spo0A strain produces all the insecticidal proteins encoded in its genome, including cry2Ab, previously considered unexpressed. A genomic analysis also revealed the presence of putative VipR-binding sequences in lepidopteran-active strains, upstream from cry genes such as cry1E, cry1F, cry9D, and cry9E. Overall, our results break the dogma on the regulation of cry1A and cry2A genes and provide evidence of sporulation-independent Cry toxin production in biopesticidal Bt strains.IMPORTANCEBacillus thuringiensis is a remarkably efficient entomopathogen due to its ability to produce various insecticidal proteins, such as Cry or Vip. This property has made it a highly effective biopesticide used worldwide. Our work modifies the paradigm of cry1 and cry2 genes being regulated solely by sporulation-specific sigma factors and thus exclusively expressed during this process. Indeed, we demonstrated that the VipR regulator controls the transcription of vip3Aa, cry2Aa, cry2Ab, cry1Ia, and the ami-cry1A operons encoded by a strain closely related to that of commercial biopesticides and specifically turns on their expression from the onset of the stationary phase, leading to the production of insecticidal crystals independently of sporulation. By providing new knowledge on the regulation of insecticidal protein genes, these findings bring new insight for the genetic improvement of Bt strains used as commercial biopesticides.

ICESan95 is a serine integrase integrative and conjugative element (ICE) that targets methyltransferase genes for integration. The mosaic ICE ICESag084 contains ICESan95 and partial fragments from two distinct genes (snf2 and hsdM), suggesting a recombination event. This study aimed to experimentally investigate the recombination of multiple ICESan95, leading to the formation of a mosaic ICE resembling ICESag084. Using conjugation assays, we tracked this evolutionary process, confirmed the structures via whole-genome sequencing, and assessed genomic stability through serial passages. Analysis showed that ICESag084 comprises an ICESan95-like ICE, a tyrosine integrase element, and a partial ICESpy009 sequence. The ICESan95-like ICE was flanked by truncated snf2 and hsdM genes, indicating ancestral cointegration of two ICESan95-like ICEs followed by excision and sequence capture. Experiments confirmed that tandem integration of ICESan95 into adjacent snf2 (from an ICESpy009-like element) and hsdM(from the tyrosine integrase element) sites facilitated recombinational excision. This event deleted an ∼36.6-kb intervening fragment, yielding an ∼86.5-kb circularized intermediate and generating an ∼81.0-kb mosaic ICE highly similar to ICESag084. The mosaic ICE was conjugatively transferable and remained stable for ≥60 generations, while the circularized ICE was lost within 20 generations. Thus, tandem integration and recombinational excision drive the emergence and spread of mosaic ICEs. This mechanism facilitates the modular assembly of multiple mobile genetic elements, including ICESpy009, ICESan95, and a tyrosine integrase element. This process creates novel resistance islands with expanded traits, providing a key pathway for long-term adaptive evolution in bacterial pathogens.

Congenital Hypopituitarism (CH) is a complex developmental disorder characterized by variable pituitary dysfunction that is often associated with midline structural abnormalities that affect the brain, eyes and face. To date, only ∼10-15% of patients have an underlying molecular basis. Next generation sequencing was conducted on a subset of CH patients with no known genetic aetiology. Human embryonic brain tissue sections were used to generate an expression profile, and a knock-out mouse model was generated using CRISPR-Cas9 gene editing and phenotypically analysed. Two novel homozygous frameshifts in CCDC149, p.Gly278* and p.Leu222*, were identified in two unrelated CH pedigrees (three patients), respectively. Patient phenotypes included growth hormone deficiency (GHD), hypogonadotropic hypogonadism, and developmental delay/autism. Severe scoliosis was present in one pedigree, with a small anterior pituitary on MRI in the other. Human embryonic CCDC149 was localised to the developing hypothalamo-pituitary region at Carnegie stages 16-23, and Ccdc149-null mice recapitulated patient phenotypes, including growth impairment and reduced fertility compared to wild-type littermates. Our study is the first to report CCDC149 variants in association with CH. Previous studies in C.elegans report CCDC149 orthologue expression in the basal bodies of ciliated neurons, supporting the possibility of impaired ciliary function as an underlying mechanism in this complex disorder.

Dog allergy is a prevalent IgE-mediated condition, with the salivary lipocalin Can f 1 accounting for the majority of dog-specific IgE reactivity in sensitized individuals. Existing strategies for managing dog allergy primarily rely on modulating host immune responses and do not address allergen production at its source. Here, we report the generation of genetically engineered dogs lacking Can f 1. Using CRISPR-Cas9 editing and somatic cell nuclear transfer, we produced two healthy beagle puppies carrying a frameshift mutation in exon 1 of the Can f 1 gene. Salivary and hair/dander analysis demonstrated the absence of Can f 1 protein in the edited dogs. Skin prick testing in a sensitized individual demonstrated robust IgE-mediated reactivity to control dog extracts but no detectable response to extracts from the edited dogs. Together, these findings demonstrate that targeted genetic knockout of the major dog allergen is compatible with canine development and can abolish the IgE-mediated allergic response, supporting the feasibility of a gene-based approach to reducing canine allergenicity.

Burkholderia includes gram-negative saprophytes, nitrogen-fixers, and species associated with nosocomial infections. Numerous strains in the Burkholderia cepacia, Burkholderia pseudomallei, Burkholderia glumae, and Burkholderia gladioli clades carry genes for phenazine (Phz) biosynthesis. Phenazines are a large class of colored, structurally diverse microbial secondary compounds with a common nitrogen-containing tricyclic core. They act as molecular signals and extracellular electron shuttles, contributing to the competitiveness of producer organisms in their natural habitats. Phenazines also undergo redox cycling, generating reactive oxygen species that suppress the growth of other organisms. The study of phenazines has largely been confined to the model opportunistic pathogen Pseudomonas aeruginosa, and many aspects of their biology in other bacterial groups remain poorly understood. In this study, we identified genes involved in phenazine production, regulation, and resistance in Burkholderia lata 383, a member of the B. cepacia complex, which produces dimethyl 4,9-dihydroxy-1,6-phenazinedicarboxylate. We subjected this strain to a transposon mutagenesis screen and characterized the transcriptomes of phenazine and quorum-sensing (QS) mutants of B. lata 383. Our results indicate that QS regulates phenazine production in Burkholderia and confirm that this cell-cell communication also controls other phenotypic traits, including biofilm formation. Analysis of transcriptome responses to phenazine methosulfate in B. lata 383 and two closely related phenazine-non-producing Burkholderia strains revealed that these organisms cope with phenazine toxicity by upregulating pathways involved in the oxidative stress response, iron-sulfur cluster biogenesis, and multidrug efflux.IMPORTANCEBurkholderia is a diverse genus comprising over 100 agriculturally, medically, and environmentally significant species. Many members of this group produce phenazines, yet the regulatory mechanisms governing phenazine biosynthesis and self-resistance remain poorly characterized. Our study sheds light on key aspects of these pathways, revealing parallels with pseudomonads in how Burkholderia species regulate and respond to these versatile, redox-active metabolites.

The MgtC virulence factor is important during the intramacrophage stage in both classical intracellular pathogens, such as Salmonella Typhimurium, and in extracellular bacteria that transiently encounter intracellular environments during infection, such as Pseudomonas aeruginosa. In these different pathogens, mgtC expression is induced in vitro by magnesium ion depletion, a condition reported to mimic the macrophage environment. Here, we developed an unstable GFP reporter system to monitor in real time the transcriptional activation of the P. aeruginosa mgtC promoter. After in vitro validation in magnesium-defined media, this reporter system allowed visualization of the mgtC promoter induction in a subset of bacteria when P. aeruginosa localized inside cultured macrophages. In addition, although rare under our experimental conditions, in vivo activation of the mgtC promoter was observed for the first time within macrophages of live, infected zebrafish larvae, a cutting-edge vertebrate model for real-time imaging. While MgtC regulation in Salmonella is mediated by the magnesium-responsive PhoPQ two-component system, its regulation in P. aeruginosa remained unknown. The use of mutant strains for two-component regulatory systems revealed that the PhoP regulator, but not by its cognate sensor PhoQ, was required to activate P. aeruginosa MgtC expression in vitro. Unexpectedly, CbrAB, a two-component system specific to Pseudomonas species, was also involved in P. aeruginosa MgtC regulation. Both PhoP and CbrB regulatory proteins were found to directly bind the mgtC promoter, supporting a dual transcriptional control. These findings reveal substantial differences in mgtC gene regulation in different bacterial pathogens, reflecting distinct strategies to drive appropriate expression of a shared virulence factor involved in macrophage adaptation.IMPORTANCEThe adaptation of bacterial pathogens to the host intracellular microenvironment requires tight and rapid regulation of specific genes, and investigating the in vivo transcriptional dynamics of such genes is a major challenge. Here, we focused on the expression of mgtC, a gene important for adaptation to the intramacrophage environment in classical intracellular pathogens, such as Salmonella Typhimurium, and bacteria with a transient intracellular lifestyle, such as Pseudomonas aeruginosa. An unstable GFP reporter system was designed to monitor the transcriptional dynamics of P. aeruginosa mgtC. The use of this reporter system in a state-of-the-art vertebrate model for live imaging, the zebrafish embryo, allowed in vivo tracking of P. aeruginosa mgtC promoter activation inside macrophages in a living host. Furthermore, the expression of P. aeruginosa mgtC was found to be regulated through a mechanism distinct from that of Salmonella MgtC, since it involves the PhoP regulatory protein, but not the PhoQ sensor, and the Pseudomonas-specific CbrAB two-component system, reflecting diverse, finely tuned strategies to control a virulence factor shared by several major human pathogens.

The assumption that synonymous mutations are fitness-neutral is central to many foundational results in the fields of genetics, genomics, evolutionary biology, and medicine. However, recent results suggest synonymous mutations have pervasive and strong fitness effects. These vigorously debated studies in non-human model systems have even suggested that the proportion of synonymous mutations and their fitness effect sizes are similar to non-synonymous mutations. To probe the fitness effect of synonymous mutations, we utilized recent advances in base editing to test 8558 potential synonymous mutations in 128 highly essential genes in human cell lines. Importantly, our library design excluded splice-proximal sites, ensuring a direct test of codon-level synonymous effects independent of splicing disruption. We find that synonymous mutations rarely have fitness effects on growth, occurring around 37.9-fold (95% CI: 22.16-81.48-fold) less frequently than missense mutations. In this experimental context, these findings demonstrate that synonymous mutations impact cellular fitness far less frequently than missense mutations. These results deviate from earlier reports of widespread synonymous fitness effects in yeast, yet they align with recent prime editing data observed in other human cell lines.

Pathogenic immune-cardiac crosstalk underlies maladaptive remodeling in chronic heart failure, yet therapies directly targeting this axis are lacking. Glycoconjugates, which are crucial for signal transduction and extracellular matrix integrity, represent an underexploited therapeutic avenue. This study sought to define the role of glycoconjugate-metabolizing enzymes at the immune-cardiac interface and evaluate their translational potential. We performed integrative analyses of bulk and single-cell RNA sequencing data from failing human and mouse hearts. Employing mouse models of pressure overload (transverse aortic constriction) and ischemia-reperfusion, we used global and mast cell (MC)-specific gene deletion, bone-marrow chimeras, and pharmacological neutralization. Mechanistic insights were gained through multiomics profiling, including RNA-seq, ATAC-seq, CUT&Tag, and proteomics. The ganglioside GD3 synthase, St8sia1, was selectively induced in cardiac MCs during pathological remodeling in both mice and humans. MC-specific or hematopoietic deletion of St8sia1 preserved ventricular function, attenuated fibrosis, and markedly reduced neutrophil and Ly6C+ monocyte recruitment after transverse aortic constriction and ischemia-reperfusion. Therapeutic neutralization of GD3 with the clinical-grade monoclonal antibody R24 improved cardiac function and diminished scar formation after ischemia-reperfusion. Mechanistically, GD3 bound specific histone variants, such as H2A.Z and H3.3C, thereby reprogramming chromatin accessibility to activate proinflammatory and profibrotic transcriptional programs in MCs. Consequently, GD3 inhibition suppressed MC degranulation, disrupted pathogenic MC-cardiomyocyte/fibroblast crosstalk, and preserved reparative macrophage populations. The MC-restricted St8sia1-GD3 axis functions as a glyco-epigenetic checkpoint driving maladaptive cardiac remodeling. Targeting this axis represents a translatable immunomodulatory strategy to prevent the progression to chronic heart failure.

The morphological transition of fungi from vegetative hyphae to thick-walled chlamydospores enhances their longevity in harsh environmental conditions. Owing to this resilience, pathogenic fungi that form chlamydospores are particularly difficult to control. Therefore, understanding the mechanisms of chlamydospore formation is critically important. Here, we show that the hyphae of the filamentous fungus Trichoderma guizhouense can differentiate into typical terminal and intercalary chlamydospores characterized by double-layered spherical or ellipsoidal cell walls with accumulated lipid bodies and nuclei. We found that during chlamydospore formation, ribosome biogenesis was gradually downregulated, indicating the entry of cells into dormancy. Comparative transcriptomic analyses across developmental stages and media identified the Gti1/Pac2 family protein CFG1 as an essential regulator, as the Δcfg1 strain failed to form chlamydospores under all inducing conditions. Lipidomic analysis showed its involvement in lipid metabolism, and mutants lacking lipid metabolism genes pdat or dgat produced fewer chlamydospores. Our work reveals the molecular mechanism of chlamydospore formation in T. guizhouense.IMPORTANCEIn fungal biology, the morphological transition from vegetative hyphae to thick-walled, lipid-rich chlamydospores represents a fundamental developmental switch into dormancy, crucial for survival under environmental stress. Understanding the regulatory mechanisms behind this process is essential for deciphering the basic principles of fungal cell differentiation and adaptation. This study employs multi-omics approaches to systematically characterize chlamydospore formation and identifies the Gti1/Pac2 family protein CFG1 as a master regulator. Functional analysis reveals that CFG1 governs this transition by directly influencing lipid metabolism-a key pathway for spore maturation and structural integrity. These findings uncover a previously unknown molecular switch in fungal development and provide new insights into how filamentous fungi coordinate metabolic reprogramming with cellular differentiation to ensure long-term survival.

Estrogen receptor-positive (ER+), HER2-negative breast cancer is the most common breast cancer subtype. While adjuvant endocrine therapy reduces recurrence risk, identifying which patients benefit from the addition of chemotherapy remains a key clinical challenge. The Oncotype DX® 21-gene Recurrence Score assay (Exact Sciences, via Genomic Health, Inc.) was developed to address this by quantifying distant recurrence risk and informing chemotherapy decisions in early-stage ER+/HER2- disease. This diagnostic profile reviews the development, validation, and clinical evidence for Oncotype DX, including findings from the TAILORx and RxPONDER prospective trials and the subsequent development of hybrid tools integrating genomic and clinicopathological data. Alternative multiparameter molecular tests (MammaPrint, Prosigna, EndoPredict, Breast Cancer Index) are summarized and compared. We review international guideline recommendations, decision impact studies, cost-effectiveness evidence, and ongoing trials. Oncotype DX has strong prognostic evidence and has meaningfully reduced chemotherapy use, though its case as a biomarker predictive of therapeutic effect from chemotherapy rests on trial designs with important limitations. Its independent prognostic contribution beyond comprehensive clinicopathological assessment requires further clarification, and cost-effectiveness varies substantially by indication and healthcare setting.

Ichang papeda (Citrus ichangensis), a wild primitive citrus species, exhibits remarkable cold tolerance, yet the transcriptional regulation of cold-responsive genes in C. ichangensis remains poorly explored. In this study, we identified the small heat shock protein (sHSP) CiHSP26.5 as a crucial member that was substantially induced under cold stress and played a positive role in cold tolerance. Furthermore, two APETALA2/Ethylene Responsive Factor (AP2/ERF) family transcription factors, CiERF023 and CiERF041, were found to directly bind to the dehydration-responsive element (DRE) motifs in the promoter of CiHSP26.5 and function as its negative and positive regulators, respectively. In addition, CiERF023 was proven to repress CiERF041 through interacting with the DRE element in the promoter. These two TFs were differentially induced during cold stress, with CiERF023 peaking at early stages and CiERF041 at later stages. Consistently, CiERF023 acts as a negative regulator, whereas CiERF041 serves as a positive regulator, of cold tolerance by regulating CiHSP26.5. Taken together, our findings reveal that CiERF023 and CiERF041 form a hierarchical transcriptional cascade that antagonistically regulates CiHSP26.5 expression under cold stress in C. ichangensis. This study provides fresh insights into the regulatory network governing HSP-mediated cold adaptation and identifies potential targets for enhancing cold tolerance in plants.

BTN3A1 has been associated with systemic lupus erythematosus (SLE) and psoriasis; however, its relationship with rheumatoid arthritis (RA) risk, particularly regarding BTN3A1 polymorphisms and RA susceptibility remains unclear. A total of 390 age- and sex-matched participants, including RA patients and healthy controls, were enrolled. Demographic, laboratory, and clinical data were collected. Five BTN3A1 polymorphisms (rs1796520, rs3857550, rs3208733, rs6912853, rs10456045) were genotyped. Allele and genotype associations between the groups were analyzed, as well as their associations with clinical and laboratory features in RA patients. Among RA patients, 79.74% were female and 20.26% were male; among controls, 84.36% were female and 15.64% were male. For rs3208733, the frequencies of the CC genotype and C allele differed significantly between RA patients and controls. For rs6912853, the frequency of the TC genotype differed significantly. For rs10456045, the frequencies of the GG, AG, and GG + AG genotypes differed significantly. Regarding clinical and laboratory associations: for rs1796520, antinuclear antibody (ANA)-positive patients showed higher frequencies of the CC + TC genotype and C allele; anti-Rib-positive patients showed a higher frequency of the C allele. RA patients carrying the CC + TC genotype had fewer swollen joints and higher levels of IL-12p70 and IFN-α than TT carriers. For rs6912853, CC + TC carriers had fewer tender joints and higher levels of C-reactive protein (CRP) and IFN-γ than TT carriers. BTN3A1 gene polymorphisms are associated with RA risk, suggesting future therapeutic exploration of BTN3A1 in RA.