Guanine Dinucleotide Research Articles

CCDC12 gene methylation in peripheral blood as a potential biomarker for breast cancer detection

Background Aberrant DNA methylation has been identified as biomarkers for breast cancer detection. Coiled-coil domain containing 12 gene (CCDC12) implicated in tumorigenesis. This study aims to investigate the potential of blood-based CCDC12 methylation for breast cancer detection. Methods DNA methylation level of CpG sites (Cytosine-phosphate Guanine dinucleotides) in CCDC12 gene was measured by mass spectrometry in 255 breast cancer patients, 155 patients with benign breast nodules and 302 healthy controls. The association between CCDC12 methylation and breast cancer risk was evaluated by logistic regression and receiver operating characteristic curve analysis. Results A total of eleven CpG sites were analyzed. The CCDC12 methylation levels were higher in breast cancer patients. Compared to the lowest tertile of methylation level in CpG_6,7, CpG_10 and CpG_11, the highest quartile was associated with 82, 91 and 95% increased breast cancer risk, respectively. The CCDC12 methylation levels were associated with estrogen receptor (ER) and human epidermal growth factor 2 (HER2) status. In ER-negative and HER2-positive (ER-/HER2+) breast cancer subtype, the combination of four sites CpG_2, CpG_5, CpG_6,7 and CpG_11 methylation levels could distinguish ER−/HER2+ breast cancer from the controls (AUC = 0.727). Conclusion The hypermethylation levels of CCDC12 in peripheral blood could be used for breast cancer detection.

Mo than meets the eye: genomic insights into molybdoenzyme diversity of Seleniivibrio woodruffii strain S4T.

Seleniivibrio woodruffii strain S4T is an obligate anaerobe belonging to the phylum Deferribacterota. It was isolated for its ability to respire selenate and was also found to respire arsenate. The high-quality draft genome of this bacterium is 2.9 Mbp, has a G+C content of 48%, 2762 predicted genes of which 2709 are protein-coding, and 53 RNA genes. An analysis of the genome focusing on the genes encoding for molybdenum-containing enzymes (molybdoenzymes) uncovered a remarkable number of genes encoding for members of the dimethylsulfoxide reductase family of proteins (DMSOR), including putative reductases for selenate and arsenate respiration, as well as genes for nitrogen fixation. Respiratory molybdoenzymes catalyze redox reactions that transfer electrons to a variety of substrates that can act as terminal electron acceptors for energy generation. Seleniivibrio woodruffii strain S4T also has essential genes for molybdate transporters and the biosynthesis of the molybdopterin guanine dinucleotide cofactors characteristic of the active centers of DMSORs. Phylogenetic analysis revealed candidate respiratory DMSORs spanning nine subfamilies encoded within the genome. Our analysis revealed the untapped potential of this interesting microorganism and expanded our knowledge of molybdoenzyme co-occurrence.

Structure of recombinant formate dehydrogenase from Methylobacterium extorquens (MeFDH1)

Formate dehydrogenase (FDH) is critical for the conversion between formate and carbon dioxide. Despite its importance, the structural complexity of FDH and difficulties in the production of the enzyme have made elucidating its unique physicochemical properties challenging. Here, we purified recombinant Methylobacterium extorquens AM1 FDH (MeFDH1) and used cryo-electron microscopy to determine its structure. We resolved a heterodimeric MeFDH1 structure at a resolution of 2.8 Å, showing a noncanonical active site and a well-embedded Fe-S redox chain relay. In particular, the tungsten bis-molybdopterin guanine dinucleotide active site showed an open configuration with a flexible C-terminal cap domain, suggesting structural and dynamic heterogeneity in the enzyme.

Redox potentials elucidate the electron transfer pathway of NAD+-dependent formate dehydrogenases

Metal-dependent, nicotine adenine dinucleotide (NAD+)-dependent formate dehydrogenases (FDHs) are complex metalloenzymes coupling biochemical transformations through intricate electron transfer pathways. Rhodobacter capsulatus FDH is a model enzyme for understanding coupled catalysis, in that reversible CO2 reduction and formate oxidation are linked to a flavin mononuclotide (FMN)-bound diaphorase module via seven iron-sulfur (FeS) clusters as a dimer of heterotetramers. Catalysis occurs at a bis-metal-binding pterin (Mo) binding two molybdopterin guanine dinucleotides (bis-MGD), a protein-based Cys residue and a participatory sulfido ligand. Insights regarding the proposed electron transfer mechanism between the bis-MGD and the FMN have been complicated by the discovery that an alternative pathway might occur via intersubunit electron transfer between two [4Fe4S] clusters within electron transfer distance. To clarify this difference, the redox potentials of the bis-MGD and the FeS clusters were determined via redox titration by EPR spectroscopy. Redox potentials for the bis-MGD cofactor and five of the seven FeS clusters could be assigned. Furthermore, substitution of the active site residue Lys295 with Ala resulted in altered enzyme kinetics, primarily due to a more negative redox potential of the A1 [4Fe4S] cluster. Finally, characterization of the monomeric FdsGBAD heterotetramer exhibited slightly decreased formate oxidation activity and similar iron-sulfur clusters reduced relative to the dimeric heterotetramer. Comparison of the measured redox potentials relative to structurally defined FeS clusters support a mechanism by which electron transfer occurs within a heterotetrameric unit, with the interfacial [4Fe4S] cluster serving as a structural component toward the integrity of the heterodimeric structure to drive efficient catalysis.

Iron limitation indirectly reduces the Escherichia coli torCAD operon expression by a reduction of molybdenum cofactor availability.

The expression of most molybdoenzymes in Escherichia coli has so far been revealed to be regulated by anaerobiosis and requires the presence of iron, based on the necessity of the transcription factor FNR to bind one [4Fe-4S] cluster. One exception is trimethylamine-N-oxide reductase encoded by the torCAD operon, which has been described to be expressed independently from FNR. In contrast to other alternative anaerobic respiratory systems, the expression of the torCAD operon was shown not to be completely repressed by the presence of dioxygen. To date, the basis for the O2-dependent expression of the torCAD operon has been related to the abundance of the transcriptional regulator IscR, which represses the transcription of torS and torT, and is more abundant under aerobic conditions than under anaerobic conditions. In this study, we reinvestigated the regulation of the torCAD operon and its dependence on the presence of iron and identified a novel regulation that depends on the presence of the bis-molybdopterin guanine dinucleotide (bis-MGD) molybdenum cofactor . We confirmed that the torCAD operon is directly regulated by the heme-containing protein TorC and is indirectly regulated by ArcA and by the availability of iron via active FNR and Fur, both regulatory proteins that influence the synthesis of the molybdenum cofactor. Furthermore, we identified a novel regulation mode of torCAD expression that is dependent on cellular levels of bis-MGD and is not used by other bis-MGD-containing enzymes like nitrate reductase.IMPORTANCEIn bacteria, molybdoenzymes are crucial for anaerobic respiration using alternative electron acceptors. FNR is a very important transcription factor that represents the master switch for the expression of target genes in response to anaerobiosis. Only Escherichia coli trimethylamine-N-oxide (TMAO) reductase escapes this regulation by FNR. We identified that the expression of TMAO reductase is regulated by the amount of bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor synthesized by the cell itself, representing a novel regulation pathway for the expression of an operon coding for a molybdoenzyme. Furthermore, TMAO reductase gene expression is indirectly regulated by the presence of iron, which is required for the production of the bis-MGD cofactor in the cell.

Development of Cupriavidus necator H16 as a host for heterologous production of formate dehydrogenase I of Methylorubrum extorquens: Possibilities and limitations

The discovery of formate dehydrogenase (Me-FDH1) from Methylorubrum extorquens has provided an avenue for sustainable CO2 fixation and utilization. However, the mass production of Me-FDH1 is challenging due to the presence of its unique tungsto-bis-metalopterin guanine dinucleotide (W-bis-MGD) cofactor, limiting its practical applications. In this study, C. necator H16 is proposed as a host for the large-scale production of Me-FDH1, utilizing fructose as a carbon source and its inherent machinery for cofactor synthesis. In a minimal salt medium, C. necator H16 could produce active Me-FDH1, which exhibited a specific activity of 80 to 100 U/mg for CO2 conversion to formate. In fed batch bioreactor experiments, approximately 50 g CDW/L (cell dry weight/L) and 10,000 U/L Me-FDH1 were achieved within 50 h. This study highlights C. necator H16 as the recombinant host for Me-FDH1, paving the way for the future development of efficient mass-production methods for this crucial enzyme.

The Reversible Electrochemical Interconversion of Formate and CO2 by Formate Dehydrogenase from Cupriavidus necator.

The bacterial molybdenum (Mo)-containing formate dehydrogenase (FdsDABG) from Cupriavidus necator is a soluble NAD+-dependent enzyme belonging to the DMSO reductase family. The holoenzyme is complex and possesses nine redox-active cofactors including a bis(molybdopterin guanine dinucleotide) (bis-MGD) active site, seven iron-sulfur clusters, and 1 equiv of flavin mononucleotide (FMN). FdsDABG catalyzes the two-electron oxidation of HCOO- (formate) to CO2 and reversibly reduces CO2 to HCOO- under physiological conditions close to its thermodynamic redox potential. Here we develop an electrocatalytically active formate oxidation/CO2 reduction system by immobilizing FdsDABG on a glassy carbon electrode in the presence of coadsorbents such as chitosan and glutaraldehyde. The reversible enzymatic interconversion between HCOO- and CO2 by FdsDABG has been realized with cyclic voltammetry using a range of artificial electron transfer mediators, with methylene blue (MB) and phenazine methosulfate (PMS) being particularly effective as electron acceptors for FdsDABG in formate oxidation. Methyl viologen (MV) acts as both an electron acceptor (MV2+) in formate oxidation and an electron donor (MV+•) for CO2 reduction. The catalytic voltammetry was reproduced by electrochemical simulation across a range of sweep rates and concentrations of formate and mediators to provide new insights into the kinetics of the FdsDABG catalytic mechanism.

The Campylobacter concisus BisA protein plays a dual role: oxide-dependent anaerobic respiration and periplasmic methionine sulfoxide repair.

Campylobacter concisus is an excellent model organism to study respiration diversity, including anaerobic respiration of physiologically relevant N-/S-oxides compounds, such as biotin sulfoxide, dimethyl sulfoxide, methionine sulfoxide (MetO), nicotinamide N-oxide, and trimethylamine N-oxide. All C. concisus strains harbor at least two, often three, and up to five genes encoding for putative periplasmic Mo/W-bisPGD-containing N-/S-oxide reductases. The respective role (substrate specificity) of each enzyme was studied using a mutagenesis approach. One of the N/SOR enzymes, annotated as "BisA", was found to be essential for anaerobic respiration of both N- and S-oxides. Additional phenotypes associated with disruption of the bisA gene included increased sensitivity toward oxidative stress and elongated cell morphology. Furthermore, a biochemical approach confirmed that BisA can repair protein-bound MetO residues. Hence, we propose that BisA plays a role as a periplasmic methionine sulfoxide reductase. This is the first report of a Mo/W-bisPGD-enzyme supporting both N- or S-oxide respiration and protein-bound MetO repair in a pathogen.

A comparison of feature selection methodologies and learning algorithms in the development of a DNA methylation-based telomere length estimator

BackgroundThe field of epigenomics holds great promise in understanding and treating disease with advances in machine learning (ML) and artificial intelligence being vitally important in this pursuit. Increasingly, research now utilises DNA methylation measures at cytosine–guanine dinucleotides (CpG) to detect disease and estimate biological traits such as aging. Given the challenge of high dimensionality of DNA methylation data, feature-selection techniques are commonly employed to reduce dimensionality and identify the most important subset of features. In this study, our aim was to test and compare a range of feature-selection methods and ML algorithms in the development of a novel DNA methylation-based telomere length (TL) estimator. We utilised both nested cross-validation and two independent test sets for the comparisons.ResultsWe found that principal component analysis in advance of elastic net regression led to the overall best performing estimator when evaluated using a nested cross-validation analysis and two independent test cohorts. This approach achieved a correlation between estimated and actual TL of 0.295 (83.4% CI [0.201, 0.384]) on the EXTEND test data set. Contrastingly, the baseline model of elastic net regression with no prior feature reduction stage performed less well in general—suggesting a prior feature-selection stage may have important utility. A previously developed TL estimator, DNAmTL, achieved a correlation of 0.216 (83.4% CI [0.118, 0.310]) on the EXTEND data. Additionally, we observed that different DNA methylation-based TL estimators, which have few common CpGs, are associated with many of the same biological entities.ConclusionsThe variance in performance across tested approaches shows that estimators are sensitive to data set heterogeneity and the development of an optimal DNA methylation-based estimator should benefit from the robust methodological approach used in this study. Moreover, our methodology which utilises a range of feature-selection approaches and ML algorithms could be applied to other biological markers and disease phenotypes, to examine their relationship with DNA methylation and predictive value.

Impact of the Dimethyl Sulfoxide Reductase Superfamily on the Evolution of Biogeochemical Cycles.

The dimethyl sulfoxide reductase (or MopB) family is a diverse assemblage of enzymes found throughout Bacteria and Archaea. Many of these enzymes are believed to have been present in the last universal common ancestor (LUCA) of all cellular lineages. However, gaps in knowledge remain about how MopB enzymes evolved and how this diversification of functions impacted global biogeochemical cycles through geologic time. In this study, we perform maximum likelihood phylogenetic analyses on manually curated comparative genomic and metagenomic data sets containing over 47,000 distinct MopB homologs. We demonstrate that these enzymes constitute a catalytically and mechanistically diverse superfamily defined not by the molybdopterin- or tungstopterin-containing [molybdopterin or tungstopterin bis(pyranopterin guanine dinucleotide) (Mo/W-bisPGD)] cofactor but rather by the structural fold that binds it in the protein. Our results suggest that major metabolic innovations were the result of the loss of the metal cofactor or the gain or loss of protein domains. Phylogenetic analyses also demonstrated that formate oxidation and CO2 reduction were the ancestral functions of the superfamily, traits that have been vertically inherited from the LUCA. Nearly all of the other families, which drive all other biogeochemical cycles mediated by this superfamily, originated in the bacterial domain. Thus, organisms from Bacteria have been the key drivers of catalytic and biogeochemical innovations within the superfamily. The relative ordination of MopB families and their associated catalytic activities emphasize fundamental mechanisms of evolution in this superfamily. Furthermore, it underscores the importance of prokaryotic adaptability in response to the transition from an anoxic to an oxidized atmosphere. IMPORTANCE The MopB superfamily constitutes a repertoire of metalloenzymes that are central to enduring mysteries in microbiology, from the origin of life and how microorganisms and biogeochemical cycles have coevolved over deep time to how anaerobic life adapted to increasing concentrations of O2 during the transition from an anoxic to an oxic world. Our work emphasizes that phylogenetic analyses can reveal how domain gain or loss events, the acquisition of novel partner subunits, and the loss of metal cofactors can stimulate novel radiations of enzymes that dramatically increase the catalytic versatility of superfamilies. We also contend that the superfamily concept in protein evolution can uncover surprising kinships between enzymes that have remarkably different catalytic and physiological functions.

Methylation of BDNF gene in association with episodic memory in women.

Brain-derived neurotrophic factor (BDNF) gene regulation plays an important role in long-term memory formation, and the DNA methylation (DNAm) level of BDNF promoters has been associated with episodic memory deficits. Our aim was to explore the association between DNAm levels in BDNF promoter IV with verbal learning and memory performance in healthy women. We conducted a cross-sectional study by recruiting 53 individuals. Episodic memory was assessed by using the Rey Auditory Verbal Learning Test (RAVLT). Clinical interviews, RAVLT, and blood sample collection were assessed in all participants. DNAm was measured on DNA from whole peripheral blood using pyrosequencing. According to generalized linear model (GzLM) analyses, cytosine guanine dinucleotide (CpG) site 5 showed significant associations between learning capacity (LC, p < 0.035), that is, every 1% of DNA methylation at CpG site 5 results in a 0.068 reduction in verbal learning performance. To the best of our knowledge, the current study is the first to show that BDNF DNAm plays an important role in episodic memory.

Genome-Wide DNA Methylation Profiling in Blastic Plasmacytoid Dendritic Cell Neoplasm

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and aggressive blood cancer and an ongoing therapeutic challenge. In a recent multiomics analysis, we characterized the unique molecular landscape of BPDCN. Beyond an expected classical pDC-derived subtype, we thereby identified a common(c)DC-enriched subtype, which was shown to predominantly arise in elderly patients, whilst harboring fewer mutations and an inferior outcome. The identification of several therapeutically targetable vulnerabilities may pave the way for precision oncology approaches toward this rare cancer. Several genomic alterations in both subtypes were found to be known determinants of the epigenetic regulation of gene expression. However, the epigenetic landscape of BPDCN remains sparsely characterized. We therefore characterized the genome-wide DNA methylation patterns in 54 BPDCN patients on the Illumina MethylationEPIC BeadChip and correlated our findings with WES/RNA-seq data from both previous and new studies. Further, we evaluated our observations according to the newly defined subtypes of BPDCN. Unsupervised clustering of the 420 most variably methylated cytosine guanine dinucleotide (CpG) sites resulted in the segregation of the study cohort into four clusters. These were subsequently annotated genomically and transcriptionally in a clinical context. Cluster two was hereby shown to be of special interest as it was exclusively composed of C2-BPDCN cases with SRSF2 or DNMT3A mutations. Cluster four revealed a strong C1-BPDCN predominance and typical C1 driver mutations including EP300, ARID2 and NF1 (Figure 1). Secondly, differential methylation analysis according to our recently established subtypes of BPDCN revealed a subset of significantly differentially methylated genes between the two clusters. Overall, we observed more methylation among C2 patients with regulation of several oncogenes and tumor suppressor genes such as NOTCH4 and MAP3K13 but a predominant regulation of inflammatory genes including TLR9, IL10RA and IL2RA. Our current observations suggest that genome-wide DNA methylation levels resemble and potentially help shape clinically and genomically distinct subgroups of BPDCN. Synoptic analysis of the methylome in concert with gene-expression profiling data alongside ATAC-Seq of our BPDCN samples is currently under way in order to integratively assess chromatin accessibility and ultimately transcriptional impact. Figure legend Figure 1. Genome-wide DNA methylation profiling in blastic plasmacytoid dendritic cell neoplasm. Unsupervised clustering of the 420 most variably methylated CpG sites resulted in the segregation of the study cohort into four clusters. These were subsequently annotated genomically and transcriptionally in a clinical context. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Potential prognostic markers of retained placenta in dairy cows identified by plasma metabolomics coupled with clinical laboratory indicators

: Background The complex etiopathology of retained placenta (RP) and hazards associated with it has made it crucial for researchers and clinical veterinarians to study pathogenesis, early-warning diagnosis, and treatment. Objectives This study aimed to screen the potential prognostic markers of RP in dairy cows using plasma metabolomics coupled with clinical laboratory indicators. Methods Blood samples were collected from 260 dairy cows at 21, 14, 7, and 0 days before parturition and 7, 14, and 21 days after parturition. Consequently, 10 healthy cows and 10 cows with RP with similar parity, body condition score, and age were included in the study. The changes in clinical laboratory indicators of the enrolled cows from 21 before parturition to 21 days after parturition were assessed. After initial overview of the multivariate statistical data using PCA analysis, the data were subjected to orthogonal partial least-squares discriminant analysis. Results Compared with cows with RP at 7 days before parturition, the levels of endothelin and 6-keto-prostaglandin F1α were increased in healthy cows while the level of estradiol and progesterone decreased. Adenine dinucleotide phosphate, hypoxanthine, guanine dinucleotide phosphate, inosine monophosphate, and L-arginine were revealed as potential prognostic markers of cows with RP at 7 days before parturition involved in the regulation of taste transduction, purine and glutathione metabolism, and autophagy. Conclusions The best period for the early-warning diagnosis of RP in dairy cows is 7 days before parturition, and purine metabolism and autophagy may play a vital role in the occurrence and development of RP in dairy cows.

Infrared Spectroscopy Elucidates the Inhibitor Binding Sites in a Metal-Dependent Formate Dehydrogenase.

Biological carbon dioxide (CO2 ) reduction is an important step by which organisms form valuable energy-richer molecules required for further metabolic processes. The Mo-dependent formate dehydrogenase (FDH) from Rhodobacter capsulatus catalyzes reversible formate oxidation to CO2 at a bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor. To elucidate potential substrate binding sites relevant for the mechanism, we studied herein the interaction with the inhibitory molecules azide and cyanate, which are isoelectronic to CO2 and charged as formate. We employed infrared (IR) spectroscopy in combination with density functional theory (DFT) and inhibition kinetics. One distinct inhibitory molecule was found to bind to either a non-competitive or a competitive binding site in the secondary coordination sphere of the active site. Site-directed mutagenesis of key amino acid residues in the vicinity of the bis-MGD cofactor revealed changes in both non-competitive and competitive binding, whereby the inhibitor is in case of the latter interaction presumably bound between the cofactor and the adjacent Arg587.

Comparison of Therapeutic Antibiotics, Probiotics, and Synthetic CpG-ODNs for Protective Efficacy Against Escherichia coli Lethal Infection and Impact on the Immune System in Neonatal Broiler Chickens.

The poultry industry needs alternatives to antibiotics, as there are growing public concerns about the emergence of antimicrobial resistance owing to antimicrobial use in animal production. We have reported that the administration of neonatal chicks with synthetic DNA oligodeoxynucleotides containing unmethylated cytosine guanine dinucleotide (CpG) motifs (CpG-ODN) can protect against bacterial pathogens in chickens. The objective of this study was to compare the immunoprotective effects of CpG-ODN and probiotics against Escherichia coli infection vs. commonly used therapeutic antibiotics. Day-old broiler chicks were divided into five groups (n = 35/group; 30 for the challenge experiment and 5 for the flow cytometry analysis). The chicks in Group 1 received a single dose of CpG-ODN by the intramuscular route on day 4 (D4) posthatch (PH), and Group 2 received drinking water (DW) with a probiotic product (D1-D15 PH, DW). The Group 3 chicks received tetracycline antibiotics during D9-D13 in DW; the Group 4 chicks got sodium sulfamethazine on D9, D10, and D15 PH in DW; and the Group 5 chicks were administered intramuscular (IM) saline D4 PH, DW. We challenged all the groups (n = 30/group) with E. coli (1 × 105 or 1 × 106 colony-forming units/bird) on D8 PH through the subcutaneous route. Our data demonstrated that the CpG-ODNs, but not the probiotics, could protect neonatal broiler chickens against lethal E. coli septicemia, as would the tetracycline or sodium sulfamethazine. The flow cytometry analysis (n = 5/group) revealed enrichment of immune cells in the CpG-ODN group and a marked decrease in macrophages and T-cell numbers in antibiotics-treated groups, indicating immunosuppressive effects. Our data showed that, like therapeutic antibiotics, CpG-ODNs reduced clinical signs, decreased bacterial loads, and induced protection in chicks against E. coli septicemia. Unlike therapeutic antibiotics-induced immunosuppressive effects, CpG-ODN caused immune enrichment by increasing chicken immune cells recruitment. Furthermore, this study highlights that, although therapeutic antibiotics can treat bacterial infections, the ensuing immunosuppressive effects may negatively impact the overall chicken health.

Enhanced systemic antilymphoma immune response by photothermal therapy with CpG deoxynucleotide–coated nanoparticles

AbstractIn preclinical studies, we investigated a novel mechanism of in situ vaccination in lymphoma. Radiation therapy (RT) can induce abscopal responses in lymphoma models, but this has not translated into clinical efficacy. We hypothesized that immune stimulation with cytosine guanine dinucleotide (CpG) deoxynucleotides could enhance abscopal effects induced by RT or photothermal therapy (PTT), which has been shown to have an immune stimulatory effect in solid tumors but has not been studied in lymphoma. We designed a branched gold nanoparticle (NP) platform to carry CpG deoxynucleotides while maintaining PTT function and compared the immunologic profile of the tumor microenvironment after PTT or RT in a dual-flank lymphoma model. One flank was treated with CpG deoxynucleotides with RT or PTT, and the other tumor was left untreated. We found that the CpG deoxynucleotide/PTT group had significant reduction in growth in both treated (primary) and untreated (secondary) tumors, suggesting an improved abscopal response, with a concomitant increase in CD8/CD4 and cytotoxic T-cell/regulatory T-cell ratios in both primary and secondary tumors compared with CpG deoxynucleotides/RT. Dendritic cells in primary and secondary draining lymph nodes had increased maturation markers in the CpG deoxynucleotide/PTT group, and the effector memory T cells (both CD4 and CD8) in the secondary tumor and spleen were increased, suggesting a systemic vaccination effect. These data suggest that in a lymphoma model, PTT using a CpG deoxynucleotide NP platform resulted in enhanced in situ vaccination and abscopal response compared with RT.

The Role of the Nucleotides in the Insertion of the bis-Molybdopterin Guanine Dinucleotide Cofactor into apo-Molybdoenzymes.

The role of the GMP nucleotides of the bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor of the DMSO reductase family has long been a subject of discussion. The recent characterization of the bis-molybdopterin (bis-Mo-MPT) cofactor present in the E. coli YdhV protein, which differs from bis-MGD solely by the absence of the nucleotides, now enables studying the role of the nucleotides of bis-MGD and bis-MPT cofactors in Moco insertion and the activity of molybdoenzymes in direct comparison. Using the well-known E. coli TMAO reductase TorA as a model enzyme for cofactor insertion, we were able to show that the GMP nucleotides of bis-MGD are crucial for the insertion of the bis-MGD cofactor into apo-TorA.

Gestational Perfluoroalkyl Substance Exposure and DNA Methylation at Birth and 12 Years of Age: A Longitudinal Epigenome-Wide Association Study.

Background:DNA methylation alterations may underlie associations between gestational perfluoroalkyl substances (PFAS) exposure and later-life health outcomes. To the best of our knowledge, no longitudinal studies have examined the associations between gestational PFAS and DNA methylation.Objectives:We examined associations of gestational PFAS exposure with longitudinal DNA methylation measures at birth and in adolescence using the Health Outcomes and Measures of the Environment (HOME) Study (2003–2006; Cincinnati, Ohio).Methods:We quantified serum concentrations of perfluorooctanoate (PFOA), perfluorooctane sulfonate (PFOS), perfluorononanoate (PFNA), and perfluorohexane sulfonate (PFHxS) in mothers during pregnancy. We measured DNA methylation in cord blood () and peripheral leukocytes at 12 years of age () using the Illumina HumanMethylation EPIC BeadChip. We analyzed associations between PFAS concentrations and repeated DNA methylation measures using linear regression with generalized estimating equations. We included interaction terms between children’s age and gestational PFAS. We performed Gene Ontology enrichment analysis to identify molecular pathways. We used Project Viva (1999–2002; Boston, Massachusetts) to replicate significant associations.Results:After adjusting for covariates, 435 cytosine–guanine dinucleotide (CpG) sites were associated with PFAS (false discovery rate, ). Specifically, we identified 2 CpGs for PFOS, 12 for PFOA, 8 for PFHxS, and 413 for PFNA; none overlapped. Among these, 2 CpGs for PFOA and 4 for PFNA were replicated in Project Viva. Some of the PFAS-associated CpG sites annotated to gene regions related to cancers, cognitive health, cardiovascular disease, and kidney function. We found little evidence that the associations between PFAS and DNA methylation differed by children’s age.Discussion:In these longitudinal data, PFAS biomarkers were associated with differences in several CpGs at birth and at 12 years of age in or near genes linked to some PFAS-associated health outcomes. Future studies should examine whether DNA methylation mediates associations between gestational PFAS exposure and health. https://doi.org/10.1289/EHP10118

Associations between alcohol use and peripheral, genetic, and epigenetic markers of oxytocin in a general sample of young and older adults.

IntroductionHuman and nonhuman animal research suggests that greater oxytocin (OT) activity is protective against harmful substance use. Most research on this topic is preclinical, with few studies evaluating the association between substance use and individual differences in the human OT system. The present study sought to fill this gap by evaluating the relationship between alcohol use and multiple biological measures of OT activity in an overall low to moderate‐drinking sample.MethodAs part of a larger study, generally healthy young (n = 51) and older (n = 53) adults self‐reported whether they regularly used alcohol and how much alcohol they consumed per week. Participants also provided blood samples from which peripheral OT, and in an age‐heterogeneous subset of participants (n = 56) variation in the oxytocin receptor gene (the OXTR rs53576 polymorphism) and OXTR DNA methylation levels (at cytosine–guanine dinucleotide sites ‐860, ‐924, ‐934), were obtained.ResultsA‐allele carriers of the OXTR rs53579 polymorphism were less likely to regularly consume alcohol. Among regular alcohol consumers, number of alcoholic drinks per week was positively associated with peripheral OT in regression models excluding observations of high influence (postdiagnostic models). Number of alcoholic drinks per week was consistently negatively associated with OXTR DNA methylation at site ‐860; and with OXTR DNA methylation at site ‐924 in postdiagnostic models.ConclusionsThe significant associations between alcohol use and individual differences in OT activity support the involvement of the OT system in alcohol use, which most likely reflect the role of OT when alcohol use is under control of its rewarding properties and/or the acute impacts of alcohol on the OT system. Additional research with markers of OT activity and alcohol use, particularly longitudinal, is needed to clarify the bidirectional effects of OT and alcohol use in moderate to harmful drinking and dependence.

Effective CpG Delivery Using Zwitterion-Functionalized Dendrimer-Entrapped Gold Nanoparticles to Promote T Cell-Mediated Immunotherapy of Cancer Cells.

Recently, cell-based immunotherapy has become one of the most promising ways to completely eliminate cancer. The major challenge is to effectively promote a proper immune response to kill the cancer cells by activated T cells. This study investigated the effect of T cell-mediated immunotherapy trigged by Au DENPs-MPC (zwitterion 2-methacryloyloxyethyl phosphorylcholine (MPC)-functionalized dendrimer-entrapped gold nanoparticles) loading oli-godeoxynucleotides (ODN) of unmethylated cytosine guanine dinucleotide (CPG). Here, we first synthesized Au DENPs-MPC, evaluated their capability to compress and transfect CpG-ODN to bone marrow dendritic cells (BMDCs), and investigated the potential to use T cells stimulated by matured BMDCs to inhibit the growth of tumor cells. The developed Au DENPs-MPC could apparently reduce the toxicity of Au DENPs, and enhanced transfer CpG-ODN to the BMDCs for the maturation as demonstrated by the 44.41–48.53% increase in different surface maturation markers. The transwell experiments certificated that ex vivo activated T cells display excellent anti-tumor ability, which could effectively inhibit the growth of tumor cells. These results suggest that Au DENPs-MPC can deliver CpG-ODN efficiently to enhance the antigen presentation ability of BMDCs to activate T cells, indicating that T cells-based immunotherapy mediated by Au DENPs-MPC loaded with CpG-ODN may become the most promising treatment of cancer.