Amnio acid substitution at position 298 of human glucose-6 phosphatase-α significantly impacts its stability in mammalian cells.

Glucose-6-phosphatase-α (G6Pase-α) catalyzes the hydrolysis of glucose-6-phosphate to glucose and functions as a key regulator in maintaining blood glucose homeostasis. Deficiency in G6Pase-α causes glycogen storage disease 1a (GSD1a), an inherited disorder characterized by life-threatening hypoglycemia and other long-term complications. We have developed a potential mRNA-based therapy for GSD1a and demonstrated that a human G6Pase-α (hG6Pase-α) variant harboring a single serine (S) to cysteine (C) substitution at the amino acid site 298 (S298C) had > twofold increase in protein expression, resulting in improved in vivo efficacy. Here, we sought to investigate the mechanisms contributing to the increased expression of the S298C variant. Mutagenesis of hG6Pase-α identified distinct protein variants at the 298 amino acid position with substantial reduction in protein expression in cultured cells. Kinetic analysis of expression and subcellular localization in mammalian cells, combined with cell-free in vitro translation assays, revealed that altered protein expression stemmed from differences in cellular protein stability rather than biosynthetic rates. Site-specific mutagenesis studies targeting other cysteines of the hG6Pase-α S298C variant suggest the observed improvements in stability are not due to additional disulfide bond formation. The glycosylation at Asparagine (N)-96 is critical in maintaining enzymatic activity and mutations at position 298 mainly affected glycosylated forms of hG6Pase-α. Finally, proteasome inhibition by lactacystin improved expression levels of unstable hG6Pase-α variants. Taken together, these data uncover a critical role for a single amino acid substitution impacting the stability of G6Pase-α and provide insights into the molecular genetics of GSD1a and protein engineering for therapeutic development.

Comprehensive Assessment of Oxidatively Induced Modifications of DNA in a Rat Model of Human Wilson's Disease.

Defective copper excretion from hepatocytes in Wilson's disease causes accumulation of copper ions with increased generation of reactive oxygen species via the Fenton-type reaction. Here we developed a nanoflow liquid chromatography-nanoelectrospray ionization-tandem mass spectrometry coupled with the isotope-dilution method for the simultaneous quantification of oxidatively induced DNA modifications. This method enabled measurement, in microgram quantities of DNA, of four oxidative stress-induced lesions, including direct ROS-induced purine cyclonucleosides (cPus) and two exocyclic adducts induced by byproducts of lipid peroxidation, i.e. 1,N(6)-etheno-2'-deoxyadenosine (εdA) and 1,N(2)-etheno-2'-deoxyguanosine (εdG). Analysis of liver tissues of Long-Evans Cinnamon rats, which constitute an animal model of human Wilson's disease, and their healthy counterparts [i.e. Long-Evans Agouti rats] showed significantly higher levels of all four DNA lesions in Long-Evans Cinnamon than Long-Evans Agouti rats. Moreover, cPus were present at much higher levels than εdA and εdG lesions. In contrast, the level of 5-hydroxymethyl-2'-deoxycytidine (5-HmdC), an oxidation product of 5-methyl-2'-deoxycytidine (5-mdC), was markedly lower in the liver tissues of Long-Evans Cinnamon than Long-Evans Agouti rats, though no differences were observed for the levels of 5-mdC. In vitro biochemical assay showed that Cu(2+) ions could directly inhibit the activity of Tet enzymes. Together, these results suggest that aberrant copper accumulation may perturb genomic stability by elevating oxidatively induced DNA lesions, and by altering epigenetic pathways of gene regulation.

Cytotoxic and Mutagenic Properties of C3'-Epimeric Lesions of 2'-Deoxyribonucleosides in Escherichia coli Cells.

Reactive oxygen species (ROS), resulting from endogenous metabolism and/or environmental exposure, can induce damage to the 2-deoxyribose moiety in DNA. Specifically, a hydrogen atom from each of the five carbon atoms in 2-deoxyribose can be abstracted by hydroxyl radical, and improper chemical repair of the ensuing radicals formed at the C1', C3', and C4' positions can lead to the stereochemical inversion at these sites to yield epimeric 2-deoxyribose lesions. Although ROS-induced single-nucleobase lesions have been well studied, the biological consequences of the C3'-epimeric lesions of 2'-deoxynucleosides, i.e., 2'-deoxyxylonucleosides (dxN), have not been comprehensively investigated. Herein, we assessed the impact of dxN lesions on the efficiency and fidelity of DNA replication in Escherichia coli cells by conducting a competitive replication and adduct bypass assay with single-stranded M13 phage containing a site-specifically incorporated dxN. Our results revealed that, of the four dxN lesions, only dxG constituted a strong impediment to DNA replication, and intriguingly, dxT and dxC conferred replication bypass efficiencies higher than those of the unmodified counterparts. In addition, the three SOS-induced DNA polymerases (Pol II, Pol IV, and Pol V) did not play any appreciable role in bypassing these lesions. Among the four dxNs, only dxA directed a moderate frequency of dCMP misincorporation. These results provided important insights into the impact of the C3'-epimeric lesions on DNA replication in E. coli cells.

Replicative Bypass Studies of α-Anomeric Lesions of 2'-Deoxyribonucleosides in Vitro.

Genomic integrity is constantly challenged by a variety of endogenous and exogenous DNA damaging agents, which can lead to the formation of 104-105 DNA lesions per cell per day. Reactive oxygen species (ROS) represent a major type of DNA damaging agent. Specifically, a hydroxyl radical can attack the C1' position of 2-deoxyribose, and the ensuing carbon-centered radical, if improperly repaired, can cause the inversion of stereochemical configuration at the C1' to give α-anomeric lesions. In this study, we assessed the replicative bypass of α-dA, α-dT, α-dC, and α-dG in template DNA by conducting primer extension assays with the use of purified translesion synthesis DNA polymerases. Our results revealed that human polymerase (Pol) η, but not human Pol κ, Pol ι, or yeast Pol ζ, was capable of bypassing all of the α-dN lesions and extending the primer to generate full-length replication products. Data from steady-state kinetic measurements showed that Pol η was the most efficient in inserting the correct nucleotides opposite the modified nucleosides, with the relative efficiencies of nucleotide incorporation following the order of α-dA > α-dG > α-dT > α-dC. Additionally, human Pol η was found to misincorporate dTMP opposite α-dT and dCMP opposite α-dC at frequencies of 66% and 24%, respectively, whereas α-dA and α-dG were weakly miscoding. These findings provided important knowledge about the effects these α-dN lesions have on the fidelity and efficiency of DNA replication mediated by human Pol η.

Cytotoxic and mutagenic properties of O4-alkylthymidine lesions in Escherichia coli cells.

Due to the abundant presence of alkylating agents in living cells and the environment, DNA alkylation is generally unavoidable. Among the alkylated DNA lesions, O(4)-alkylthymidine (O(4)-alkyldT) are known to be highly mutagenic and persistent in mammalian tissues. Not much is known about how the structures of the alkyl group affect the repair and replicative bypass of the O(4)-alkyldT lesions, or how the latter process is modulated by translesion synthesis polymerases. Herein, we synthesized oligodeoxyribonucleotides harboring eight site-specifically inserted O(4)-alkyldT lesions and examined their impact on DNA replication in Escherichia coli cells. We showed that the replication past all the O(4)-alkyldT lesions except (S)- and (R)-sBudT was highly efficient, and these lesions directed very high frequencies of dGMP misincorporation in E. coli cells. While SOS-induced DNA polymerases play redundant roles in bypassing most of the O(4)-alkyldT lesions, the bypass of (S)- and (R)-sBudT necessitated Pol V. Moreover, Ada was not involved in the repair of any O(4)-alkyldT lesions, Ogt was able to repair O(4)-MedT and, to a lesser extent, O(4)-EtdT and O(4)-nPrdT, but not other O(4)-alkyldT lesions. Together, our study provided important new knowledge about the repair of the O(4)-alkyldT lesions and their recognition by the E. coli replication machinery.

In vivo detection and replication studies of α-anomeric lesions of 2'-deoxyribonucleosides.

DNA damage, arising from endogenous metabolism or exposure to environmental agents, may perturb the transmission of genetic information by blocking DNA replication and/or inducing mutations, which contribute to the development of cancer and likely other human diseases. Hydroxyl radical attack on the C1', C3' and C4' of 2-deoxyribose can give rise to epimeric 2-deoxyribose lesions, for which the in vivo occurrence and biological consequences remain largely unexplored. Through independent chemical syntheses of all three epimeric lesions of 2'-deoxyguanosine (dG) and liquid chromatography-tandem mass spectrometry analysis, we demonstrated unambiguously the presence of substantial levels of the α-anomer of dG (α-dG) in calf thymus DNA and in DNA isolated from mouse pancreatic tissues. We further assessed quantitatively the impact of all four α-dN lesions on DNA replication in Escherichia coli by employing a shuttle-vector method. We found that, without SOS induction, all α-dN lesions except α-dA strongly blocked DNA replication and, while replication across α-dA was error-free, replicative bypass of α-dC and α-dG yielded mainly C→A and G→A mutations. In addition, SOS induction could lead to markedly elevated bypass efficiencies for the four α-dN lesions, abolished the G→A mutation for α-dG, pronouncedly reduced the C→A mutation for α-dC and triggered T→A mutation for α-dT. The preferential misincorporation of dTMP opposite the α-dNs could be attributed to the unique base-pairing properties of the nucleobases elicited by the inversion of the configuration of the N-glycosidic linkage. Our results also revealed that Pol V played a major role in bypassing α-dC, α-dG and α-dT in vivo. The abundance of α-dG in mammalian tissue and the impact of the α-dNs on DNA replication demonstrate for the first time the biological significance of this family of DNA lesions.

Simultaneous Quantification of Methylated Cytidine and Adenosine in Cellular and Tissue RNA by Nano-Flow Liquid Chromatography-Tandem Mass Spectrometry Coupled with the Stable Isotope-Dilution Method.

The rising interest in understanding the functions, regulation, and maintenance of the epitranscriptome calls for robust and accurate analytical methods for the identification and quantification of post-transcriptionally modified nucleosides in RNA. Monomethylations of cytidine and adenosine are common post-transcriptional modifications in RNA. Herein, we developed an LC-MS/MS/MS coupled with the stable isotope-dilution method for the sensitive and accurate quantifications of 5-methylcytidine (m(5)C), 2'-O-methylcytidine (Cm), N(6)-methyladenosine (m(6)A), and 2'-O-methyladenosine (Am) in RNA isolated from mammalian cells and tissues. Our results showed that the distributions of m(5)C, Cm and Am are tissue-specific. In addition, the 2'-O-methylated ribonucleosides (Cm and Am) are present at higher levels than the corresponding methylated nucleobase products (m(5)C and m(6)A) in total RNA isolated from mouse brain, pancreas, and spleen but not mouse heart. We also found that the levels of m(5)C, Cm, and Am are significantly lower (by 6.5-43-fold) in mRNA than in total RNA isolated from HEK293T cells, whereas the level of m(6)A was slightly higher (by 1.6-fold) in mRNA than in total RNA. The availability of this analytical method, in combination with genetic manipulation, may facilitate the future discovery of proteins involved in the maintenance and regulation of these RNA modifications.

Photochemical Generation of a C5'-Uridinyl Radical.

It has been postulated that sugar radicals and related species are involved in oxidative events involving RNA. To determine the contribution, if any, of these species to the deleterious effects of the endogenous exposome, it is important to unambiguously identify their degradation products. C5'-Pivaloyl uridine was successfully synthesized and subsequently photolytically converted to a C5'-uridinyl radical. Generation of the radical under anaerobic conditions in the presence of glutathione led to the formation of the expected reduction product, uridine. However, regardless of the presence or absence of reductant, the base elimination product, uracil, was also observed. Mass balances and product distributions were dependent upon the pH of the photolysis mixture. At low pH, trapping with glutathione successfully competed with base loss. These results indicate that this precursor should function efficiently in an investigation of the fate of the C5'-uridinyl radical in RNA oligomers.

Arsenite Targets the Zinc Finger Domains of Tet Proteins and Inhibits Tet-Mediated Oxidation of 5-Methylcytosine.

Arsenic toxicity is a serious public health problem worldwide that brings more than 100 million people into the risk of arsenic exposure from groundwater and food contamination. Although there is accumulating evidence linking arsenic exposure with aberrant cytosine methylation in the global genome or at specific genomic loci, very few have investigated the impact of arsenic on the oxidation of 5-methylcytosine (5-mC) mediated by the Ten-eleven translocation (Tet) family of proteins. Owing to the high binding affinity of As(III) toward cysteine residues, we reasoned that the highly conserved C3H-type zinc fingers situated in Tet proteins may constitute potential targets for arsenic binding. Herein, we found that arsenite could bind directly to the zinc fingers of Tet proteins in vitro and in cells, and this interaction substantially impaired the catalytic efficiency of Tet proteins in oxidizing 5-mC to 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-foC), and 5-carboxylcytosine (5-caC). Treatments with arsenite also led to a dose-dependent decrease in the level of 5-hmC, but not 5-mC, in DNA isolated from HEK293T cells overexpressing the catalytic domain of any of the three Tet proteins and from mouse embryonic stem cells. Together, our study unveiled, for the first time, that arsenite could alter epigenetic signaling by targeting the zinc fingers of Tet proteins and perturbing the Tet-mediated oxidation of 5-mC in vitro and in cells. Our results offer important mechanistic understanding of arsenic epigenotoxicity and carcinogenesis in mammalian systems and may lead to novel approaches for the chemoprevention of arsenic toxicity.

Tet-mediated formation of 5-hydroxymethylcytosine in RNA.

Oxidation of 5-methylcytosine in DNA by ten-eleven translocation (Tet) family of enzymes has been demonstrated to play a significant role in epigenetic regulation in mammals. We found that Tet enzymes also possess the activity of catalyzing the formation of 5-hydroxymethylcytidine (5-hmrC) in RNA in vitro. In addition, the catalytic domains of all three Tet enzymes as well as full-length Tet3 could induce the formation of 5-hmrC in human cells. Moreover, 5-hmrC was present at appreciable levels (∼1 per 5000 5-methylcytidine) in RNA of mammalian cells and tissues. Our results suggest the involvement of this oxidation in RNA biology.

Role of the androgen receptor in breast cancer and preclinical analysis of enzalutamide.

INTRODUCTION: The androgen receptor (AR) is widely expressed in breast cancers and has been proposed as a therapeutic target in estrogen receptor alpha (ER) negative breast cancers that retain AR. However, controversy exists regarding the role of AR, particularly in ER + tumors. Enzalutamide, an AR inhibitor that impairs nuclear localization of AR, was used to elucidate the role of AR in preclinical models of ER positive and negative breast cancer. METHODS: We examined nuclear AR to ER protein ratios in primary breast cancers in relation to response to endocrine therapy. The effects of AR inhibition with enzalutamide were examined in vitro and in preclinical models of ER positive and negative breast cancer that express AR. RESULTS: In a cohort of 192 women with ER + breast cancers, a high ratio of AR:ER (≥2.0) indicated an over four fold increased risk for failure while on tamoxifen (HR = 4.43). The AR:ER ratio had an independent effect on risk for failure above ER % staining alone. AR:ER ratio is also an independent predictor of disease-free survival (HR = 4.04, 95% CI: 1.68, 9.69; p = 0.002) and disease specific survival (HR = 2.75, 95% CI: 1.11, 6.86; p = 0.03). Both enzalutamide and bicalutamide inhibited 5-alpha-dihydrotestosterone (DHT)-mediated proliferation of breast cancer lines in vitro; however, enzalutamide uniquely inhibited estradiol (E2)-mediated proliferation of ER+/AR + breast cancer cells. In MCF7 xenografts (ER+/AR+) enzalutamide inhibited E2-driven tumor growth as effectively as tamoxifen by decreasing proliferation. Enzalutamide also inhibited DHT- driven tumor growth in both ER positive (MCF7) and negative (MDA-MB-453) xenografts, but did so by increasing apoptosis. CONCLUSIONS: AR to ER ratio may influence breast cancer response to traditional endocrine therapy. Enzalutamide elicits different effects on E2-mediated breast cancer cell proliferation than bicalutamide. This preclinical study supports the initiation of clinical studies evaluating enzalutamide for treatment of AR+ tumors regardless of ER status, since it blocks both androgen- and estrogen- mediated tumor growth.

Epimeric 2-deoxyribose lesions: Products from the improper chemical repair of 2-deoxyribose radicals.

Genomic integrity is constantly challenged by DNA damaging agents such as reactive oxygen species (ROS). Consequently, DNA damage can compromise the fidelity and efficiency of essential DNA metabolic processes, including replication and transcription, which may contribute significantly to the etiology of many human diseases. Here, we review one family of DNA lesions, the epimeric 2-deoxyribose lesions, which arise from the improper chemical repair of the 2-deoxyribose radicals. Unlike most other DNA lesions, the epimeric 2-deoxyribose lesions are indistinguishable from their corresponding unmodified nucleosides in both molecular mass and chemical reactivity. We placed our emphasis of discussion on the formation of these lesions, their impact on the structure and stability of duplex DNA, their biological consequences, their potential therapeutic relevance, and future research directions about these modified nucleosides.

The impact of structure on oxidatively generated DNA damage products resulting from the C3'-thymidinyl radical.

What's the damage? Trapping the C3'-thymidinyl radical in biologically significant architectures delivers both the repaired oligomer and 1-(2'-deoxy-ß-D-threo-pentofuranosyl)thymidine-containing substrates. The stereoselectivity of the reduction was found to be dependent upon the DNA structure.

A series of N-(2-phenylethyl)nitroaniline derivatives as precursors for slow and sustained nitric oxide release agents.

2,4-Dinitro-N-(2-phenylethyl)aniline, C14H13N3O4, (I), crystallizes with one independent molecule in the asymmetric unit. The adjacent amine and nitro groups form an intramolecular N-H...O hydrogen bond. The anti conformation about the ethyl C-C bond leads to the phenyl and aniline rings being essentially parallel. Molecules are linked into dimers by intermolecular N-H...O hydrogen bonds, such that each amine H atom participates in a three-centre interaction with two nitro O atoms. Though the dimers pack so that the arene rings of adjacent molecules are parallel, the rings are staggered and π-π interactions do not appear to be favoured. 4,6-Dinitro-N,N'-bis(2-phenylethyl)benzene-1,3-diamine, C22H22N4O4, (II), differs from (I) in the presence of a second 2-phenylethylamine group on the substituted ring. Compound (II) also crystallizes with one unique molecule in the asymmetric unit. Both amine groups are involved in intramolecular N-H...O hydrogen bonds with adjacent nitro groups. Although one ethyl group adopts an anti conformation as in (I), the other is gauche, with the result that the pendant phenyl rings are not parallel. The amine group that is part of the gauche conformation participates in a three-centre N-H...O hydrogen bond with the nitro group of a neighbouring molecule, leading to dimers as in (I). The other amine H atom does not form any intermolecular hydrogen bonds. The packing leads to separations of ca 3.4 Šof the parallel anti phenyl and aminobenzene rings. 2-Cyano-4-nitro-N-(2-phenylethyl)aniline, C15H13N3O2, (III), differs from (I) only in having a cyano group in place of the 2-nitro group. The absence of the adjacent nitro group eliminates the intramolecular N-H...O hydrogen bond. Molecules of (III) adopt the same anti conformation about the ethyl group as in (I), but crystallize in the higher-symmetry monoclinic space group P21/n. The molecules are linked into dimers via N-H...N amine-cyano hydrogen bonds, while the nitro groups are not involved in any N-H...O interactions. Owing to the different symmetry, the molecules pack in a herringbone pattern with fewer face-to-face interactions between the rings. The closest such interactions are about 3.5 Šbetween rings that are largely slipped past one another. 4-Methylsulfonyl-2-nitro-N-(2-phenylethyl)aniline, C15H16N2O4S, (IV), differs from (I) in having a methylsulfonyl group in place of the 4-nitro group. The intramolecular N-H...O hydrogen bond is present as in (I). However, unlike (I), the conformation about the ethyl group is gauche, so the two arene rings are nearly perpendicular rather than parallel. The packing is significantly different from the other three structures in that there are no intermolecular hydrogen bonds involving the N-H groups. The molecules are arranged in tetragonal columns running along the c axis, with the aniline rings mostly parallel and separated by ca 3.7 Å. Taken together, these structures demonstrate that modest changes in functional groups cause significant differences in molecular conformation, intermolecular interactions and packing.

Bivalent SARS-CoV-2 mRNA vaccines increase breadth of neutralization and protect against the BA.5 Omicron variant in mice.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants in the Omicron lineage has resulted in diminished Coronavirus Disease 2019 (COVID-19) vaccine efficacy and persistent transmission. In this study, we evaluated the immunogenicity and protective efficacy of two, recently authorized, bivalent COVID-19 vaccines that contain two mRNAs encoding Wuhan-1 and either BA.1 (mRNA-1273.214) or BA.4/5 (mRNA-1273.222) spike proteins. As a primary two-dose immunization series in mice, both bivalent vaccines induced greater neutralizing antibody responses against Omicron variants than the parental, monovalent mRNA-1273 vaccine. When administered to mice as a booster at 7 months after the primary vaccination series with mRNA-1273, the bivalent vaccines induced broadly neutralizing antibody responses. Whereas most anti-Omicron receptor binding domain antibodies in serum induced by mRNA-1273, mRNA-1273.214 and mRNA-1273.222 boosters cross-reacted with the antecedent Wuhan-1 spike antigen, the mRNA-1273.214 and mRNA-1273.222 bivalent vaccine boosters also induced unique BA.1-specific and BA.4/5-specific responses, respectively. Although boosting with parental or bivalent mRNA vaccines substantially improved protection against BA.5 compared to mice receiving two vaccine doses, the levels of infection, inflammation and pathology in the lung were lowest in animals administered the bivalent mRNA vaccines. Thus, boosting with bivalent Omicron-based mRNA-1273.214 or mRNA-1273.222 vaccines enhances immunogenicity and confers protection in mice against a currently circulating SARS-CoV-2 strain.

Neutrophil functional heterogeneity is a fixed phenotype and is associated with distinct gene expression profiles.

Differences in the ability of neutrophils to perform relevant effector functions has been identified in a variety of disease states. Although neutrophil functional heterogeneity is increasingly recognized during disease, few studies have examined neutrophil functional heterogeneity during periods of health. In this study, we systematically characterize neutrophil functional heterogeneity in a cohort of healthy human subjects using a range of biologically relevant agonists including immune complexes, bacterial ligands, and pathogens. With repeated testing over several years, we show that neutrophil functional capability represents a fixed phenotype for each individual. This neutrophil phenotype is preserved across a range of agonists and extends to a variety of effector functions including degranulation, neutrophil extracellular trap release, reactive oxygen species generation, phagocytosis, and bacterial killing. Using well-phenotyped healthy human subjects, we demonstrate that neutrophil functional heterogeneity is characterized by differences in neutrophil gene expression patterns. Altogether, our findings demonstrate that while neutrophil function is highly heterogeneous among healthy subjects, each individual's functional capability represents a fixed phenotype defined by a distinct neutrophil gene expression profile. These findings may be relevant during disease states where the ability to perform relevant neutrophil effector functions may impact disease course and/or clinical outcome.

Altering the mRNA-1273 dosing interval impacts the kinetics, quality, and magnitude of immune responses in mice.

For a vaccine to achieve durable immunity and optimal efficacy, many require a multi-dose primary vaccination schedule that acts to first "prime" naive immune systems and then "boost" initial immune responses by repeated immunizations (ie, prime-boost regimens). In the context of the global coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), 2-dose primary vaccination regimens were often selected with short intervals between doses to provide rapid protection while still inducing robust immunity. However, emerging post-authorization evidence has suggested that longer intervals between doses 1 and 2 for SARS-CoV-2 vaccines may positively impact robustness and durability of immune responses. Here, the dosing interval for mRNA-1273, a messenger RNA based SARS-CoV-2 vaccine administered on a 2-dose primary schedule with 4 weeks between doses, was evaluated in mice by varying the dose interval between 1 and 8 weeks and examining immune responses through 24 weeks after dose 2. A dosing interval of 6 to 8 weeks generated the highest level of antigen-specific serum immunoglobulin G binding antibody titers. Differences in binding antibody titers between mRNA-1273 1 µg and 10 µg decreased over time for dosing intervals of ≥4 weeks, suggesting a potential dose-sparing effect. Longer intervals (≥4 weeks) also increased antibody-dependent cellular cytotoxicity activity and numbers of antibody-secreting cells (including long-lived plasma cells) after the second dose. An interval of 6 to 8 weeks elicited the strongest CD8+ T-cell responses, while an interval of 3 weeks elicited the strongest CD4+ T-cell response. Overall, these results suggest that in a non-pandemic setting, a longer interval (≥6 weeks) between the doses of the primary series for mRNA-1273 may induce more durable immune responses.

Bivalent SARS-CoV-2 mRNA vaccines increase breadth of neutralization and protect against the BA.5 Omicron variant.

The emergence of SARS-CoV-2 variants in the Omicron lineage with large numbers of substitutions in the spike protein that can evade antibody neutralization has resulted in diminished vaccine efficacy and persistent transmission. One strategy to broaden vaccine-induced immunity is to administer bivalent vaccines that encode for spike proteins from both historical and newly-emerged variant strains. Here, we evaluated the immunogenicity and protective efficacy of two bivalent vaccines that recently were authorized for use in Europe and the United States and contain two mRNAs encoding Wuhan-1 and either BA.1 (mRNA-1273.214) or BA.4/5 (mRNA-1273.222) spike proteins. As a primary immunization series in BALB/c mice, both bivalent vaccines induced broader neutralizing antibody responses than the constituent monovalent vaccines (mRNA-1273 [Wuhan-1], mRNA-1273.529 [BA.1], and mRNA-1273-045 [BA.4/5]). When administered to K18-hACE2 transgenic mice as a booster at 7 months after the primary vaccination series with mRNA-1273, the bivalent vaccines induced greater breadth and magnitude of neutralizing antibodies compared to an mRNA-1273 booster. Moreover, the response in bivalent vaccine-boosted mice was associated with increased protection against BA.5 infection and inflammation in the lung. Thus, boosting with bivalent Omicron-based mRNA-1273.214 or mRNA-1273.222 vaccines enhances immunogenicity and protection against currently circulating SARS-CoV-2 strains.

CpG island tumor suppressor promoter methylation in non-BRCA-associated early mammary carcinogenesis.

BACKGROUND: Only 5% of all breast cancers are the result of BRCA1/2 mutations. Methylation silencing of tumor suppressor genes is well described in sporadic breast cancer; however, its role in familial breast cancer is not known. METHODS: CpG island promoter methylation was tested in the initial random periareolar fine-needle aspiration sample from 109 asymptomatic women at high risk for breast cancer. Promoter methylation targets included RARB (M3 and M4), ESR1, INK4a/ARF, BRCA1, PRA, PRB, RASSF1A, HIN-1, and CRBP1. RESULTS: Although the overall frequency of CpG island promoter methylation events increased with age (P<0.0001), no specific methylation event was associated with age. In contrast, CpG island methylation of RARB M4 (P=0.051), INK4a/ARF (P=0.042), HIN-1 (P=0.044), and PRA (P=0.032), as well as the overall frequency of methylation events (P=0.004), was associated with abnormal Masood cytology. The association between promoter methylation and familial breast cancer was tested in 40 unaffected premenopausal women in our cohort who underwent BRCA1/2 mutation testing. Women with BRCA1/2 mutations had a low frequency of CpG island promoter methylation (15 of 15 women had

Cytotoxic and Mutagenic Properties of C1' and C3'-Epimeric Lesions of 2'-Deoxyribonucleosides in Human Cells.

Genomic integrity is constantly challenged by exposure to environmental and endogenous genotoxic agents. Reactive oxygen species (ROS) represent one of the most common types of DNA damaging agents. While ROS mainly induce single-nucleobase lesions, epimeric 2-deoxyribose lesions can also be induced upon hydrogen atom abstraction from the C1', C3', or C4' carbon and the subsequent incorrect chemical repair of the resulting carbon-centered radicals. Herein, we investigated the replicative bypass of the C1'- and C3'-epimeric lesions of the four 2'-deoxynucleosides in HEK293T human embryonic kidney epithelial cells. Our results revealed distinct bypass efficiencies and mutagenic properties of these two types of epimeric lesions. Replicative bypasses of all C1'-epimeric lesions except α-dA are mutagenic in HEK293T cells, and their mutagenic properties are further modulated by translesion synthesis (TLS) DNA polymerases. By contrast, none of the four C3'-epimeric lesions are mutagenic, and the replicative bypass of these lesions is not compromised upon depletion of polymerase η, ι, κ, or ζ. Together, our results provide important new knowledge about the cytotoxic and mutagenic properties of C1' and C3' epimeric lesions, and reveal the roles of TLS DNA polymerases in bypassing these lesions in human cells.