Necroptosis blockade prevents lung injury in severe influenza.

Severe influenza A virus (IAV) infections can result in hyper-inflammation, lung injury and acute respiratory distress syndrome1-5 (ARDS), for which there are no effective pharmacological therapies. Necroptosis is an attractive entry point for therapeutic intervention in ARDS and related inflammatory conditions because it drives pathogenic lung inflammation and lethality during severe IAV infection6-8 and can potentially be targeted by receptor interacting protein kinase 3 (RIPK3) inhibitors. Here we show that a newly developed RIPK3 inhibitor, UH15-38, potently and selectively blocked IAV-triggered necroptosis in alveolar epithelial cells in vivo. UH15-38 ameliorated lung inflammation and prevented mortality following infection with laboratory-adapted and pandemic strains of IAV, without compromising antiviral adaptive immune responses or impeding viral clearance. UH15-38 displayed robust therapeutic efficacy even when administered late in the course of infection, suggesting that RIPK3 blockade may provide clinical benefit in patients with IAV-driven ARDS and other hyper-inflammatory pathologies.

Adaptive foraging behaviours in the Horn of Africa during Toba supereruption.

Although modern humans left Africa multiple times over 100,000 years ago, those broadly ancestral to non-Africans dispersed less than 100,000 years ago1. Most models hold that these events occurred through green corridors created during humid periods because arid intervals constrained population movements2. Here we report an archaeological site-Shinfa-Metema 1, in the lowlands of northwest Ethiopia, with Youngest Toba Tuff cryptotephra dated to around 74,000 years ago-that provides early and rare evidence of intensive riverine-based foraging aided by the likely adoption of the bow and arrow. The diet included a wide range of terrestrial and aquatic animals. Stable oxygen isotopes from fossil mammal teeth and ostrich eggshell show that the site was occupied during a period of high seasonal aridity. The unusual abundance of fish suggests that capture occurred in the ever smaller and shallower waterholes of a seasonal river during a long dry season, revealing flexible adaptations to challenging climatic conditions during the Middle Stone Age. Adaptive foraging along dry-season waterholes would have transformed seasonal rivers into 'blue highway' corridors, potentially facilitating an out-of-Africa dispersal and suggesting that the event was not restricted to times of humid climates. The behavioural flexibility required to survive seasonally arid conditions in general, and the apparent short-term effects of the Toba supereruption in particular were probably key to the most recent dispersal and subsequent worldwide expansion of modern humans.

CRISPR-mediated adaptive immune systems in bacteria and archaea.

Effective clearance of an infection requires that the immune system rapidly detects and neutralizes invading parasites while strictly avoiding self-antigens that would result in autoimmunity. The cellular machinery and complex signaling pathways that coordinate an effective immune response have generally been considered properties of the eukaryotic immune system. However, a surprisingly sophisticated adaptive immune system that relies on small RNAs for sequence-specific targeting of foreign nucleic acids was recently discovered in bacteria and archaea. Molecular vaccination in prokaryotes is achieved by integrating short fragments of foreign nucleic acids into a repetitive locus in the host chromosome known as a CRISPR (clustered regularly interspaced short palindromic repeat). Here we review the mechanisms of CRISPR-mediated immunity and discuss the ecological and evolutionary implications of these adaptive defense systems.

Variants in ZFX are associated with an X-linked neurodevelopmental disorder with recurrent facial gestalt.

Pathogenic variants in multiple genes on the X chromosome have been implicated in syndromic and non-syndromic intellectual disability disorders. ZFX on Xp22.11 encodes a transcription factor that has been linked to diverse processes including oncogenesis and development, but germline variants have not been characterized in association with disease. Here, we present clinical and molecular characterization of 18 individuals with germline ZFX variants. Exome or genome sequencing revealed 11 variants in 18 subjects (14 males and 4 females) from 16 unrelated families. Four missense variants were identified in 11 subjects, with seven truncation variants in the remaining individuals. Clinical findings included developmental delay/intellectual disability, behavioral abnormalities, hypotonia, and congenital anomalies. Overlapping and recurrent facial features were identified in all subjects, including thickening and medial broadening of eyebrows, variations in the shape of the face, external eye abnormalities, smooth and/or long philtrum, and ear abnormalities. Hyperparathyroidism was found in four families with missense variants, and enrichment of different tumor types was observed. In molecular studies, DNA-binding domain variants elicited differential expression of a small set of target genes relative to wild-type ZFX in cultured cells, suggesting a gain or loss of transcriptional activity. Additionally, a zebrafish model of ZFX loss displayed an altered behavioral phenotype, providing additional evidence for the functional significance of ZFX. Our clinical and experimental data support that variants in ZFX are associated with an X-linked intellectual disability syndrome characterized by a recurrent facial gestalt, neurocognitive and behavioral abnormalities, and an increased risk for congenital anomalies and hyperparathyroidism.

Structure Reveals a Mechanism of CRISPR-RNA-Guided Nuclease Recruitment and Anti-CRISPR Viral Mimicry.

Bacteria and archaea have evolved sophisticated adaptive immune systems that rely on CRISPR RNA (crRNA)-guided detection and nuclease-mediated elimination of invading nucleic acids. Here, we present the cryo-electron microscopy (cryo-EM) structure of the type I-F crRNA-guided surveillance complex (Csy complex) from Pseudomonas aeruginosa bound to a double-stranded DNA target. Comparison of this structure to previously determined structures of this complex reveals a ∼180-degree rotation of the C-terminal helical bundle on the "large" Cas8f subunit. We show that the double-stranded DNA (dsDNA)-induced conformational change in Cas8f exposes a Cas2/3 "nuclease recruitment helix" that is structurally homologous to a virally encoded anti-CRISPR protein (AcrIF3). Structural homology between Cas8f and AcrIF3 suggests that AcrIF3 is a mimic of the Cas8f nuclease recruitment helix.

Strategic vision for improving human health at The Forefront of Genomics.

Starting with the launch of the Human Genome Project three decades ago, and continuing after its completion in 2003, genomics has progressively come to have a central and catalytic role in basic and translational research. In addition, studies increasingly demonstrate how genomic information can be effectively used in clinical care. In the future, the anticipated advances in technology development, biological insights, and clinical applications (among others) will lead to more widespread integration of genomics into almost all areas of biomedical research, the adoption of genomics into mainstream medical and public-health practices, and an increasing relevance of genomics for everyday life. On behalf of the research community, the National Human Genome Research Institute recently completed a multi-year process of strategic engagement to identify future research priorities and opportunities in human genomics, with an emphasis on health applications. Here we describe the highest-priority elements envisioned for the cutting-edge of human genomics going forward-that is, at 'The Forefront of Genomics'.

Author Correction: Rubber bands reduce the cost of carrying loads.

In this Brief Communication, the scale bar in Fig. 2a should be '0.1 m' rather than '0.01 m'. This has not been corrected online.

The Candida albicans Cdk8-dependent phosphoproteome reveals repression of hyphal growth through a Flo8-dependent pathway.

Ssn3, also known as Cdk8, is a member of the four protein Cdk8 submodule within the multi-subunit Mediator complex involved in the co-regulation of transcription. In Candida albicans, the loss of Ssn3 kinase activity affects multiple phenotypes including cellular morphology, metabolism, nutrient acquisition, immune cell interactions, and drug resistance. In these studies, we generated a strain in which Ssn3 was replaced with a functional variant of Ssn3 that can be rapidly and selectively inhibited by the ATP analog 3-MB-PP1. Consistent with ssn3 null mutant and kinase dead phenotypes, inhibition of Ssn3 kinase activity promoted hypha formation. Furthermore, the increased expression of hypha-specific genes was the strongest transcriptional signal upon inhibition of Ssn3 in transcriptomics analyses. Rapid inactivation of Ssn3 was used for phosphoproteomic studies performed to identify Ssn3 kinase substrates associated with filamentation potential. Both previously validated and novel Ssn3 targets were identified. Protein phosphorylation sites that were reduced specifically upon Ssn3 inhibition included two sites in Flo8 which is a transcription factor known to positively regulate C. albicans morphology. Mutation of the two Flo8 phosphosites (threonine 589 and serine 620) was sufficient to increase Flo8-HA levels and Flo8 dependent transcriptional and morphological changes, suggesting that Ssn3 kinase activity negatively regulates Flo8.Under embedded conditions, when ssn3Δ/Δ and efg1Δ/Δ mutants were hyperfilamentous, FLO8 was essential for hypha formation. Previous work has also shown that loss of Ssn3 activity leads to increased alkalinization of medium with amino acids. Here, we show that the ssn3Δ/Δ medium alkalinization phenotype, which is dependent on STP2, a transcription factor involved in amino acid utilization, also requires FLO8 and EFG1. Together, these data show that Ssn3 activity can modulate Flo8 and its direct and indirect interactions in different ways, and underscores the potential importance of considering Ssn3 function in the control of transcription factor activities.

Computational engineering of previously crystallized pyruvate formate-lyase activating enzyme reveals insights into SAM binding and reductive cleavage.

Radical S-adenosyl-l-methionine (SAM) enzymes are ubiquitous in nature and carry out a broad variety of difficult chemical transformations initiated by hydrogen atom abstraction. Although numerous radical SAM (RS) enzymes have been structurally characterized, many prove recalcitrant to crystallization needed for atomic-level structure determination using X-ray crystallography, and even those that have been crystallized for an initial study can be difficult to recrystallize for further structural work. We present here a method for computationally engineering previously observed crystallographic contacts and employ it to obtain more reproducible crystallization of the RS enzyme pyruvate formate-lyase activating enzyme (PFL-AE). We show that the computationally engineered variant binds a typical RS [4Fe-4S]2+/+ cluster that binds SAM, with electron paramagnetic resonance properties indistinguishable from the native PFL-AE. The variant also retains the typical PFL-AE catalytic activity, as evidenced by the characteristic glycyl radical electron paramagnetic resonance signal observed upon incubation of the PFL-AE variant with reducing agent, SAM, and PFL. The PFL-AE variant was also crystallized in the [4Fe-4S]2+ state with SAM bound, providing a new high-resolution structure of the SAM complex in the absence of substrate. Finally, by incubating such a crystal in a solution of sodium dithionite, the reductive cleavage of SAM is triggered, providing us with a structure in which the SAM cleavage products 5'-deoxyadenosine and methionine are bound in the active site. We propose that the methods described herein may be useful in the structural characterization of other difficult-to-resolve proteins.

Electronic Health Record-Based Algorithm for Monitoring Respiratory Virus-Like Illness.

Viral respiratory illness surveillance has traditionally focused on single pathogens (e.g., influenza) and required fever to identify influenza-like illness (ILI). We developed an automated system applying both laboratory test and syndrome criteria to electronic health records from 3 practice groups in Massachusetts, USA, to monitor trends in respiratory viral-like illness (RAVIOLI) across multiple pathogens. We identified RAVIOLI syndrome using diagnosis codes associated with respiratory viral testing or positive respiratory viral assays or fever. After retrospectively applying RAVIOLI criteria to electronic health records, we observed annual winter peaks during 2015-2019, predominantly caused by influenza, followed by cyclic peaks corresponding to SARS-CoV-2 surges during 2020-2024, spikes in RSV in mid-2021 and late 2022, and recrudescent influenza in late 2022 and 2023. RAVIOLI rates were higher and fluctuations more pronounced compared with traditional ILI surveillance. RAVIOLI broadens the scope, granularity, sensitivity, and specificity of respiratory viral illness surveillance compared with traditional ILI surveillance.

Evolving use of ancestry, ethnicity, and race in genetics research-A survey spanning seven decades.

To inform continuous and rigorous reflection about the description of human populations in genomics research, this study investigates the historical and contemporary use of the terms "ancestry," "ethnicity," "race," and other population labels in The American Journal of Human Genetics from 1949 to 2018. We characterize these terms' frequency of use and assess their odds of co-occurrence with a set of social and genetic topical terms. Throughout The Journal's 70-year history, "ancestry" and "ethnicity" have increased in use, appearing in 33% and 26% of articles in 2009-2018, while the use of "race" has decreased, occurring in 4% of articles in 2009-2018. Although its overall use has declined, the odds of "race" appearing in the presence of "ethnicity" has increased relative to the odds of occurring in its absence. Forms of population descriptors "Caucasian" and "Negro" have largely disappeared from The Journal (<1% of articles in 2009-2018). Conversely, the continental labels "African," "Asian," and "European" have increased in use and appear in 18%, 14%, and 42% of articles from 2009-2018, respectively. Decreasing uses of the terms "race," "Caucasian," and "Negro" are indicative of a transition away from the field's history of explicitly biological race science; at the same time, the increasing use of "ancestry," "ethnicity," and continental labels should serve to motivate ongoing reflection as the terminology used to describe genetic variation continues to evolve.

Ifit2 restricts murine coronavirus spread to the spinal cord white matter and its associated myelin pathology.

Interferon-induced protein with tetratricopeptide repeats 2, Ifit2, is critical in restricting neurotropic murine-ß-coronavirus, RSA59 infection. RSA59 intracranial injection of Ifit2-deficient (-/-) compared to wild-type (WT) mice results in impaired acute microglial activation, reduced CX3CR1 expression, limited migration of peripheral lymphocytes into the brain, and impaired virus control followed by severe morbidity and mortality. While the protective role of Ifit2 is established for acute viral encephalitis, less is known about its influence during the chronic demyelinating phase of RSA59 infection. To understand this, RSA59 infected Ifit2-/- and Ifit2+/+ (WT) were observed for neuropathological outcomes at day 5 (acute phase) and 30 post-infection (chronic phase). Our study demonstrates that Ifit2 deficiency causes extensive RSA59 spread throughout the spinal cord gray and white matter, associated with impaired CD4+ T and CD8+ T cell infiltration. Further, the cervical lymph nodes of RSA59 infected Ifit2-/- mice showed reduced activation of CD4+ T cells and impaired IFNγ expression during acute encephalomyelitis. Interestingly, BBB integrity was better preserved in Ifit2-/- mice, as evidenced by tight junction protein Claudin-5 and adapter protein ZO-1 expression surrounding the meninges and blood vessels and decreased Texas red dye uptake, which may be responsible for reduced leukocyte infiltration. In contrast to sparse myelin loss in WT mice, the chronic disease phase in Ifit2-/- mice was associated with severe demyelination and persistent viral load, even at low inoculation doses. Overall, our study highlights that Ifit2 provides antiviral functions by promoting acute neuroinflammation and thereby aiding virus control and limiting severe chronic demyelination. IMPORTANCE Interferons execute their function by inducing specific genes collectively termed as interferon-stimulated genes (ISGs), among which interferon-induced protein with tetratricopeptide repeats 2, Ifit2, is known for restricting neurotropic viral replication and spread. However, little is known about its role in viral spread to the spinal cord and its associated myelin pathology. Toward this, our study using a neurotropic murine ß-coronavirus and Ifit2-deficient mice demonstrates that Ifit2 deficiency causes extensive viral spread throughout the gray and white matter of the spinal cord accompanied by impaired microglial activation and T cell infiltration. Furthermore, infected Ifit2-deficient mice showed impaired activation of T cells in the cervical lymph node and relatively intact blood-brain barrier integrity. Overall, Ifit2 plays a crucial role in mounting host immunity against neurotropic murine coronavirus in the acute phase while preventing mice from developing viral-induced severe chronic neuroinflammatory demyelination, the characteristic feature of human neurological disease multiple sclerosis (MS).

Do Phosphodiesterase Type 5 Inhibitors Increase the Risk of Biochemical Recurrence After Radical Prostatectomy?

PURPOSE: There have been conflicting studies on the association between phosphodiesterase type 5 inhibitor (PDE5i) use and biochemical recurrence (BCR) following radical prostatectomy (RP). Our aim was to determine whether PDE5i drug exposure after RP increases the risk of BCR in patients undergoing RP. MATERIALS AND METHODS: An institutional database of prostate cancer patients treated between January 2009 and December 2020 was reviewed. BCR was defined as 2 PSA measurements greater than 0.1 ng/mL. PDE5i exposure was defined using a 0 to 3 scale, with 0 representing never use, 1 sometimes use, 2 regularly use, and 3 routinely use. The risk of BCR with any PDE5i exposure, the quantity of exposure, and the duration of PDE5i exposure were assessed by multivariable Cox proportional hazards models. RESULTS: The sample size included 4630 patients to be analyzed, with 776 patients having BCR. The median follow-up for patients without BCR was 27 (IQR 12, 49) months. Eighty-nine percent reported taking a PDE5i at any time during the first 12 months after RP, and 60% reported doing so for 6 or more months during the year after RP. There was no evidence of an increase in the risk of BCR associated with any PDE5i use (HR 1.05, 95% CI 0.84, 1.31, P = .7) or duration of PDE5i use in the first year (HR 0.98 per 1 month duration, 95% CI 0.96, 1.00, P = .055). Baseline oncologic risk was lower in patients using PDE5i, but differences between groups were small, suggesting that residual confounding is unlikely to obscure any causal association with BCR. CONCLUSIONS: Prescription of PDE5i to men after RP can be based exclusively on quality of life considerations. Patients receiving PDE5is can be reassured that their use does not increase the risk of BCR.

CUREs for high-level Galectin-3 expression.

Galectins are a large and diverse protein family defined by the presence of a carbohydrate recognition domain (CRD) that binds ß-galactosides. They play important roles in early development, tissue regeneration, immune homeostasis, pathogen recognition, and cancer. In many cases, studies that examine galectin biology and the effect of manipulating galectins are aided by, or require the ability to express and purify, specific members of the galectin family. In many cases, E. coli is employed as a heterologous expression system, and galectin expression is induced with isopropyl ß-galactoside (IPTG). Here, we show that galectin-3 recognizes IPTG with micromolar affinity and that as IPTG induces expression, newly synthesized galectin can bind and sequester cytosolic IPTG, potentially repressing further expression. To circumvent this putative inhibitory feedback loop, we utilized an autoinduction protocol that lacks IPTG, leading to significantly increased yields of galectin-3. Much of this work was done within the context of a course-based undergraduate research experience, indicating the ease and reproducibility of the resulting expression and purification protocols.

Does climate impact inflatable penile prosthesis infection (IPP) risk? Assessment of temperature and dew point on IPP infections.

BACKGROUND: Variations in climate have been associated with a greater risk of surgical site infections, urinary tract infections, and changes in the skin microbiome; however, limited data exist on the impact of climate on inflatable penile prosthesis (IPP) infections. AIM: We sought to evaluate the impact of climate on the risk of IPP infections in a large international, multicenter cohort. METHODS: We performed a multi-institutional, retrospective study of patients undergoing IPP surgery. We then evaluated whether the month or season, during which surgery was performed, affected device infections. Implant infections were defined as infections requiring device explantation. A univariate logistic regression analysis was undertaken. OUTCOMES: Our primary outcome was implant infection. RESULTS: A total of 5289 patients with a mean age of 62.2 ± 10.8 years received IPP placement. There was a fairly even distribution of implants performed in each season. A total of 103 (1.9%) infections were recorded. There were 32 (31.1%) IPP infections in patients who underwent surgery in the summer, followed by 28 (27.2%) in the winter, 26 (25.2%) in the spring, and 17 (16.5%) in the fall. No statistically significant differences were recorded in terms of season (P = .19) and month (P = .29). The mean daily temperature (P = .43), dew point (P = .43), and humidity (P = .92) at the time of IPP placement was not associated with infection. CLINICAL IMPLICATIONS: These findings provide reassurance to prosthetic urologists that infection reduction strategies do not need to be tailored to local climate. STRENGTHS AND LIMITATIONS: Climate data were not directly recorded for each hospital, but rather based on the monthly averages in the city where the surgery was performed. CONCLUSION: The climate at time of IPP placement and time of year of surgery is not associated with IPP infection risk.

Prioritizing diversity in human genomics research.

Recent studies have highlighted the imperatives of including diverse and under-represented individuals in human genomics research and the striking gaps in attaining that inclusion. With its multidecade experience in supporting research and policy efforts in human genomics, the National Human Genome Research Institute is committed to establishing foundational approaches to study the role of genomic variation in health and disease that include diverse populations. Large-scale efforts to understand biology and health have yielded key scientific findings, lessons and recommendations on how to increase diversity in genomic research studies and the genomic research workforce. Increased attention to diversity will increase the accuracy, utility and acceptability of using genomic information for clinical care.

Gestational exposure to organochlorine compounds and metals and infant birth weight: effect modification by maternal hardships.

BACKGROUND: Gestational exposure to toxic environmental chemicals and maternal social hardships are individually associated with impaired fetal growth, but it is unclear whether the effects of environmental chemical exposure on infant birth weight are modified by maternal hardships. METHODS: We used data from the Maternal-Infant Research on Environmental Chemicals (MIREC) Study, a pan-Canadian cohort of 1982 pregnant females enrolled between 2008 and 2011. We quantified eleven environmental chemical concentrations from two chemical classes - six organochlorine compounds (OCs) and five metals - that were detected in ≥ 70% of blood samples collected during the first trimester. We examined fetal growth using birth weight adjusted for gestational age and assessed nine maternal hardships by questionnaire. Each maternal hardship variable was dichotomized to indicate whether the females experienced the hardship. In our analysis, we used elastic net to select the environmental chemicals, maternal hardships, and 2-way interactions between maternal hardships and environmental chemicals that were most predictive of birth weight. Next, we obtained effect estimates using multiple linear regression, and plotted the relationships by hardship status for visual interpretation. RESULTS: Elastic net selected trans-nonachlor, lead, low educational status, racially minoritized background, and low supplemental folic acid intake. All were inversely associated with birth weight. Elastic net also selected interaction terms. Among those with increasing environmental chemical exposures and reported hardships, we observed stronger negative associations and a few positive associations. For example, every two-fold increase in lead concentrations was more strongly associated with reduced infant birth weight among participants with low educational status (ß = -100 g (g); 95% confidence interval (CI): -215, 16), than those with higher educational status (ß = -34 g; 95% CI: -63, -3). In contrast, every two-fold increase in mercury concentrations was associated with slightly higher birth weight among participants with low educational status (ß = 23 g; 95% CI: -25, 71) compared to those with higher educational status (ß = -9 g; 95% CI: -24, 6). CONCLUSIONS: Our findings suggest that maternal hardships can modify the associations of gestational exposure to some OCs and metals with infant birth weight.

A combinatorial system to examine the enzymatic repair of multiply damaged DNA substrates.

DNA damage drives genetic mutations that underlie the development of cancer in humans. Multiple pathways have been described in mammalian cells which can repair this damage. However, most work to date has focused upon single lesions in DNA. We present here a combinatorial system which allows assembly of duplexes containing single or multiple types of damage by ligating together six oligonucleotides containing damaged or modified bases. The combinatorial system has dual fluorescent labels allowing examination of both strands simultaneously, in order to study interactions or competition between different DNA repair pathways. Using this system, we demonstrate how repair of oxidative damage in one DNA strand can convert a mispaired T:G deamination intermediate into a T:A mutation. We also demonstrate that slow repair of a T:G mispair, relative to a U:G mispair, by the human methyl-binding domain 4 DNA glycosylase provides a competitive advantage to competing repair pathways, and could explain why CpG dinucleotides are hotspots for C to T mutations in human tumors. Data is also presented that suggests repair of closely spaced lesions in opposing strands can be repaired by a combination of short and long-patch base excision repair and simultaneous repair of multiply damage sites can potentially lead to lethal double strand breaks.

Sex-Related Differences in Shoulder Complex Joint Dynamics Variability During Pediatric Manual Wheelchair Propulsion.

More than 80% of adult manual wheelchair users with spinal cord injuries will experience shoulder pain. Females and those with decreased shoulder dynamics variability are more likely to experience pain in adulthood. Sex-related differences in shoulder dynamics variability during pediatric manual wheelchair propulsion may influence the lifetime risk of pain. We evaluated the influence of sex on 3-dimensional shoulder complex joint dynamics variability in 25 (12 females and 13 males) pediatric manual wheelchair users with spinal cord injury. Within-subject variability was quantified using the coefficient of variation. Permutation tests evaluated sex-related differences in variability using an adjusted critical alpha of P = .001. No sex-related differences in sternoclavicular or acromioclavicular joint kinematics or glenohumeral joint dynamics variability were observed (all P ≥ .042). Variability in motion, forces, and moments are considered important components of healthy joint function, as reduced variability may increase the likelihood of repetitive strain injury and pain. While further work is needed to generalize our results to other manual wheelchair user populations across the life span, our findings suggest that sex does not influence joint dynamics variability in pediatric manual wheelchair users with spinal cord injury.

Measurement of deaminated cytosine adducts in DNA using a novel hybrid thymine DNA glycosylase.

The hydrolytic deamination of cytosine and 5-methylcytosine drives many of the transition mutations observed in human cancer. The deamination-induced mutagenic intermediates include either uracil or thymine adducts mispaired with guanine. While a substantial array of methods exist to measure other types of DNA adducts, the cytosine deamination adducts pose unusual analytical problems, and adequate methods to measure them have not yet been developed. We describe here a novel hybrid thymine DNA glycosylase (TDG) that is comprised of a 29-amino acid sequence from human TDG linked to the catalytic domain of a thymine glycosylase found in an archaeal thermophilic bacterium. Using defined-sequence oligonucleotides, we show that hybrid TDG has robust mispair-selective activity against deaminated U:G and T:G mispairs. We have further developed a method for separating glycosylase-released free bases from oligonucleotides and DNA followed by GC-MS/MS quantification. Using this approach, we have measured for the first time the levels of total uracil, U:G, and T:G pairs in calf thymus DNA. The method presented here will allow the measurement of the formation, persistence, and repair of a biologically important class of deaminated cytosine adducts.