Base excision repair of the N-(2-deoxy-d-erythro-pentofuranosyl)-urea lesion by the hNEIL1 glycosylase.

The N-(2-deoxy-d-erythro-pentofuranosyl)-urea DNA lesion forms following hydrolytic fragmentation of cis-5R,6S- and trans-5R,6R-dihydroxy-5,6-dihydrothymidine (thymine glycol, Tg) or from oxidation of 7,8-dihydro-8-oxo-deoxyguanosine (8-oxodG) and subsequent hydrolysis. It interconverts between α and ß deoxyribose anomers. Synthetic oligodeoxynucleotides containing this adduct are efficiently incised by unedited (K242) and edited (R242) forms of the hNEIL1 glycosylase. The structure of a complex between the active site unedited mutant CΔ100 P2G hNEIL1 (K242) glycosylase and double-stranded (ds) DNA containing a urea lesion reveals a pre-cleavage intermediate, in which the Gly2 N-terminal amine forms a conjugate with the deoxyribose C1' of the lesion, with the urea moiety remaining intact. This structure supports a proposed catalytic mechanism in which Glu3-mediated protonation of O4' facilitates attack at deoxyribose C1'. The deoxyribose is in the ring-opened configuration with the O4' oxygen protonated. The electron density of Lys242 suggests the 'residue 242-in conformation' associated with catalysis. This complex likely arises because the proton transfer steps involving Glu6 and Lys242 are hindered due to Glu6-mediated H-bonding with the Gly2 and the urea lesion. Consistent with crystallographic data, biochemical analyses show that the CΔ100 P2G hNEIL1 (K242) glycosylase exhibits a residual activity against urea-containing dsDNA.

Mass Spectrometry-Based Method to Measure Aflatoxin B1 DNA Adducts in Formalin-Fixed Paraffin-Embedded Tissues.

Aflatoxin B1 (AFB1) is a potent human liver carcinogen produced by certain molds, particularly Aspergillus flavus and Aspergillus parasiticus, which contaminate peanuts, corn, rice, cottonseed, and ground and tree nuts, principally in warm and humid climates. AFB1 undergoes bioactivation in the liver to produce AFB1-exo-8,9-epoxide, which forms the covalently bound cationic AFB1-N7-guanine (AFB1-N7-Gua) DNA adduct. This adduct is unstable and undergoes base-catalyzed opening of the guanine imidazolium ring to form two ring-opened diastereomeric 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl-formamido)-9-hydroxy-aflatoxin B1 (AFB1-FapyGua) adducts. The AFB1 formamidopyrimidine (Fapy) adducts induce G → T transversion mutations and are likely responsible for the carcinogenic effects of AFB1. Quantitative liquid chromatography-mass spectrometry (LC-MS) methods have shown that AFB1-N7-Gua is eliminated in rodent and human urine, whereas ring-opened AFB1-FapyGua adducts persist in rodent liver. However, fresh frozen biopsy tissues are seldom available for biomonitoring AFB1 DNA adducts in humans, impeding research advances in this potent liver carcinogen. In contrast, formalin-fixed paraffin-embedded (FFPE) specimens used for histopathological analysis are often accessible for molecular studies. However, ensuring nucleic acid quality presents a challenge due to incomplete reversal of formalin-mediated DNA cross-links, which can preclude accurate quantitative measurements of DNA adducts. In this study, employing ion trap or high-resolution accurate Orbitrap mass spectrometry, we demonstrate that ring-opened AFB1-FapyGua adducts formed in AFB1-exposed newborn mice are stable to the formalin fixation and DNA de-cross-linking retrieval processes. The AFB1-FapyGua adducts can be detected at levels comparable to those in a match of fresh frozen liver. Orbitrap MS2 measurements can detect AFB1-FapyGua at a quantification limit of 4.0 adducts per 108 bases when only 0.8 µg of DNA is assayed on the column. Thus, our breakthrough DNA retrieval technology can be adapted to screen for AFB1 DNA adducts in FFPE human liver specimens from cohorts at risk of this potent liver carcinogen.

Understanding desiccation tolerance and sex-specific responses in Bryum argenteum: insights from phenological phases and physiological analyses.

BACKGROUND AND AIMS: Desiccation tolerance (DT) is crucial for survival in arid environments, where organisms develop strategies in reproduction, maintenance and defence to cope with water scarcity. Therefore, investigating the relationship between reproduction and DT is essential to understand the ecology and adaptive strategies of species. This study explores the connection between the development of male and female gametangia in the moss Bryum argenteum and the decrease in DT during the progression of phenological phases in gametangia and protonema. METHODS: Samples collected from a dry tropical forest in Brazil were cultivated, cloned and subjected to desiccation. Subsequently, the physiological parameters of shoots and protonemata were analysed. Shoot and protonema regeneration were monitored for 28 d after the physiological analyses. Both phases were subjected to control and desiccation treatments. KEY RESULTS: Significant effects of desiccation and sex on the physiological parameters and regeneration capacity of shoots and protonemata were found. Male shoots generally exhibited lower values of Fv/Fm (quantum efficiency of photosystem II) and ϕPSII (effective quantum yield of photosystem II), while females demonstrated higher values and better recovery after desiccation. Protonemata also showed variation in Fv/Fm over time and with sex, with no significant differences in ϕPSII between them. Desiccated male shoots had higher mortality rates and produced fewer new shoots. For females, the regeneration patterns varied between the desiccation-exposed groups and the control, with decreased shoot production, and some protonemata growing into filaments without forming shoots. CONCLUSION: These findings improve our understanding of the ecological responses of bryophytes to desiccation stress and provide insights into their adaptive strategies in challenging environments, such as the possible rarity of males in dioicous moss populations.

Enhanced cytarabine-induced killing in OGG1-deficient acute myeloid leukemia cells.

Human clinical trials suggest that inhibition of enzymes in the DNA base excision repair (BER) pathway, such as PARP1 and APE1, can be useful in anticancer strategies when combined with certain DNA-damaging agents or tumor-specific genetic deficiencies. There is also evidence suggesting that inhibition of the BER enzyme 8-oxoguanine DNA glycosylase-1 (OGG1), which initiates repair of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (Fapy-dG), could be useful in treating certain cancers. Specifically, in acute myeloid leukemia (AML), both the RUNX1-RUNX1T1 fusion and the CBFB-MYH11 subtypes have lower levels of OGG1 expression, which correlate with increased therapeutic-induced cell cytotoxicity and good prognosis for improved, relapse-free survival compared with other AML patients. Here we present data demonstrating that AML cell lines deficient in OGG1 have enhanced sensitivity to cytarabine (cytosine arabinoside [Ara-C]) relative to OGG1-proficient cells. This enhanced cytotoxicity correlated with endogenous oxidatively-induced DNA damage and Ara-C-induced DNA strand breaks, with a large proportion of these breaks occurring at common fragile sites. This lethality was highly specific for Ara-C treatment of AML cells deficient in OGG1, with no other replication stress-inducing agents showing a correlation between cell killing and low OGG1 levels. The mechanism for this preferential toxicity was addressed using in vitro replication assays in which DNA polymerase δ was shown to insert Ara-C opposite 8-oxo-dG, resulting in termination of DNA synthesis. Overall, these data suggest that incorporation of Ara-C opposite unrepaired 8-oxo-dG may be the fundamental mechanism conferring selective toxicity and therapeutic effectiveness in OGG1-deficient AML cells.

Relationships among sporophytic and gametophytic traits of 27 subtropical montane moss species.

PREMISE: Moss sporophytes differ strongly in size and biomass partitioning, potentially reflecting reproductive and dispersal strategies. Understanding how sporophyte traits are coordinated is essential for understanding moss functioning and evolution. This study aimed to answer: (1) how the size and proportions of the sporophyte differ between moss species with and without a prominent central strand in the seta, (2) how anatomical and morphological traits of the seta are related, and (3) how sporophytic biomass relates to gametophytic biomass and nutrient concentrations. METHODS: We studied the relationships between seta anatomical and morphological traits, the biomass of seta, capsule, and gametophyte, and carbon, nitrogen, and phosphorus concentrations of 27 subtropical montane moss species. RESULTS: (1) Moss species with a prominent central strand in the seta had larger setae and heavier capsules than those without a prominent strand. (2) With increasing seta length, setae became thicker and more rounded for both groups, while in species with a prominent central strand, the ratio of transport-cell area to epidermal area decreased. (3) In both groups, mosses with greater gametophytic biomass tended to have heavier sporophytes, but nitrogen and phosphorus concentrations in the gametophyte were unrelated to sporophytic traits. CONCLUSIONS: Our study highlights that the central strand in the seta may have an important functional role and affect the allometry of moss sporophytes. The coordinated variations in sporophyte morphological and anatomical traits follow basic biomechanical principles of cylinder-like structures, and these traits relate only weakly to the gametophytic nutrient concentrations. Research on moss sporophyte functional traits and their relationships to gametophytes is still in its infancy but could provide important insights into their adaptative strategies.

To dry perchance to live: Insights from the genome of the desiccation-tolerant biocrust moss Syntrichia caninervis.

With global climate change, water scarcity threatens whole agro/ecosystems. The desert moss Syntrichia caninervis, an extremophile, offers novel insights into surviving desiccation and heat. The sequenced S. caninervis genome consists of 13 chromosomes containing 16 545 protein-coding genes and 2666 unplaced scaffolds. Syntenic relationships within the S. caninervis and Physcomitrella patens genomes indicate the S. caninervis genome has undergone a single whole genome duplication event (compared to two for P. patens) and evidence suggests chromosomal or segmental losses in the evolutionary history of S. caninervis. The genome contains a large sex chromosome composed primarily of repetitive sequences with a large number of Copia and Gypsy elements. Orthogroup analyses revealed an expansion of ELIP genes encoding proteins important in photoprotection. The transcriptomic response to desiccation identified four structural clusters of novel genes. The genomic resources established for this extremophile offer new perspectives for understanding the evolution of desiccation tolerance in plants.

Self-Perception of Transition Readiness Amongst Pediatric Patients with Childhood Heart Disease.

This study assesses self-report of transition readiness among adolescents and young adults (ages 12-25 years) with childhood and congenital heart disease (CHD), receiving care at Hasbro Children's Hospital, whose CHD diagnosis warranted transfer to adult cardiology care. Patients were mailed the American Academy of Pediatrics/American College of Physicians Transition Readiness for Youth Assessment survey. Confidence scores ranged between 0 (not) and 10 (very). Mann-Whitney U test was used to assess differences in scores between younger (12-17 years) and older (18-25 years) groups. 396 patients met inclusion criteria; 88 surveys were returned. Half of respondents were in the older group. While most respondents felt empowered to take charge of their own health and equally confident about moving to adult care, this did not always translate to actual knowledge. Younger patients had statistically significant lower knowledge scores in these metrics. Aspects of care with low scores include medication refills, communication with primary care team, and the lifelong need for cardiology follow up and health insurance. This discrepancy between self-report and actual knowledge highlights the need for more varied and age appropriate interventions to help patients navigate a complex healthcare system. A concrete approach to acquire the knowledge necessary to ensure successful transfer to adult cardiology care should be a focus.

Co-Occurring Substance Use Disorders and Grief during Recovery.

BACKGROUND: Individuals with substance use disorders (SUDs) often experience loss and grief when recovering from addiction. With fatal overdoses and suicide rates increasing for individuals with SUDs and other mental health concerns, individuals in recovery are often faced with mourning the loss of a loved one while navigating their life in recovery. The loss of a loved one can be compounded by the effect of losing their relationship with their drug of choice. These co-occurring losses may prove overwhelming during recovery and precipitate a relapse. OBJECTIVES: We wish to propose the use of two bereavement models to address the grief present for individuals with SUDs experiencing loss so that providers are better able meet the complex needs of this population. METHODS: This article presents two separate bereavement models that may be used to shape treatment for individuals in recovery from addiction with co-occurring grief from the loss of a loved one: the dual-process model of coping with bereavement (DPM) and the two-track model of bereavement. RESULTS: We propose the application of these two grief models in treatment for individuals with SUDs who are experiencing co-occurring grief issues. We discuss therapeutic interventions that may be utilized to support clients with co-occurring losses.Conclusion/Importance: If an individual experiences the loss of a loved one while navigating their life in recovery from a SUD, they can experience complicated grief due to co-occurring losses. These models provide an opportunity for mental health providers to help those with SUDs work through this complex grief.

The transition of WRRF models to digital twin applications.

Digital Twins (DTs) are on the rise as innovative, powerful technologies to harness the power of digitalisation in the WRRF sector. The lack of consensus and understanding when it comes to the definition, perceived benefits and technological needs of DTs is hampering their widespread development and application. Transitioning from traditional WRRF modelling practice into DT applications raises a number of important questions: When is a model's predictive power acceptable for a DT? Which modelling frameworks are most suited for DT applications? Which data structures are needed to efficiently feed data to a DT? How do we keep the DT up to date and relevant? Who will be the main users of DTs and how to get them involved? How do DTs push the water sector to evolve? This paper provides an overview of the state-of-the-art, challenges, good practices, development needs and transformative capacity of DTs for WRRF applications.

Natural ultraviolet radiation exposure alters photosynthetic biology and improves recovery from desiccation in a desert moss.

Plants in dryland ecosystems experience extreme daily and seasonal fluctuations in light, temperature, and water availability. We used an in situ field experiment to uncover the effects of natural and reduced levels of ultraviolet radiation (UV) on maximum PSII quantum efficiency (Fv/Fm), relative abundance of photosynthetic pigments and antioxidants, and the transcriptome in the desiccation-tolerant desert moss Syntrichia caninervis. We tested the hypotheses that: (i) S. caninervis plants undergo sustained thermal quenching of light [non-photochemical quenching (NPQ)] while desiccated and after rehydration; (ii) a reduction of UV will result in improved recovery of Fv/Fm; but (iii) 1 year of UV removal will de-harden plants and increase vulnerability to UV damage, indicated by a reduction in Fv/Fm. All field-collected plants had extremely low Fv/Fm after initial rehydration but recovered over 8 d in lab-simulated winter conditions. UV-filtered plants had lower Fv/Fm during recovery, higher concentrations of photoprotective pigments and antioxidants such as zeaxanthin and tocopherols, and lower concentrations of neoxanthin and Chl b than plants exposed to near natural UV levels. Field-grown S. caninervis underwent sustained NPQ that took days to relax and for efficient photosynthesis to resume. Reduction of solar UV radiation adversely affected recovery of Fv/Fm following rehydration.

DNA Sequence Modulates the Efficiency of NEIL1-Catalyzed Excision of the Aflatoxin B1-Induced Formamidopyrimidine Guanine Adduct.

Dietary exposure to aflatoxins is a significant risk factor in the development of hepatocellular carcinomas. Following bioactivation by microsomal P450s, the reaction of aflatoxin B1 (AFB1) with guanine (Gua) in DNA leads to the formation of stable, imidazole ring-opened 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl-formamido)-9-hydroxyaflatoxin B1 (AFB1-FapyGua) adducts. In contrast to most base modifications that result in destabilization of the DNA duplex, the AFB1-FapyGua adduct increases the thermal stability of DNA via 5'-interface intercalation and base-stacking interactions. Although it was anticipated that this stabilization might make these lesions difficult to repair relative to helix distorting modifications, prior studies have shown that both the nucleotide and base excision repair pathways participate in the removal of the AFB1-FapyGua adduct. Specifically for base excision repair, we previously showed that the DNA glycosylase NEIL1 excises AFB1-FapyGua and catalyzes strand scission in both synthetic oligodeoxynucleotides and liver DNA of exposed mice. Since it is anticipated that error-prone replication bypass of unrepaired AFB1-FapyGua adducts contributes to cellular transformation and carcinogenesis, the structural and thermodynamic parameters that modulate the efficiencies of these repair pathways are of considerable interest. We hypothesized that the DNA sequence context in which the AFB1-FapyGua adduct is formed might modulate duplex stability and consequently alter the efficiencies of NEIL1-initiated repair. To address this hypothesis, site-specific AFB1-FapyGua adducts were synthesized in three sequence contexts, with the 5' neighbor nucleotide being varied. DNA structural stability analyses were conducted using UV absorbance- and NMR-based melting experiments. These data revealed differentials in thermal stabilities associated with the 5'-neighbor base pair. Single turnover kinetic analyses using the NEIL1 glycosylase demonstrated corresponding sequence-dependent differences in the repair of this adduct, such that there was an inverse correlation between the stabilization of the duplex and the efficiency of NEIL1-mediated catalysis.

Strategies of desiccation tolerance vary across life phases in the moss Syntrichia caninervis.

PREMISE: Desiccation tolerance (DT) is a widespread phenomenon among land plants, and variable ecological strategies for DT are likely to exist. Using Syntrichia caninervis, a dryland moss and model system used in DT studies, we hypothesized that DT is lowest in juvenile (protonemal) tissues, highest in asexual reproductive propagules (gemmae), and intermediate in adults (shoots). We tested the long-standing hypothesis of an inherent constitutive strategy of DT in this species. METHODS: Plants were rapidly dried to levels of equilibrating relative humidity (RHeq) ranging from 0 to 93%. Postrehydration recovery was assessed using chlorophyll fluorescence, regeneration rates, and visual tissue damage. For each life phase, we estimated the minimum rate of drying (RoDmin ) at RHeq = 42% that did not elicit damage 24 h postrehydration. RESULTS: DT strategy varied with life phase, with adult shoots having the lowest RoDmin (10-25 min), followed by gemmae (3-10 h) and protonema (14-20 h). Adult shoots exhibited no detectable damage 24 h postrehydration following a rapid-dry only at the highest RHeq used (93%), but when dried to lower RHs the response declined to <50% of control fluorescence values. Notably, immediately following rehydration (0 h postrehydration), shoots were damaged below control levels of fluorescence regardless of the RHeq, thus implicating damage. CONCLUSIONS: Life phases of the moss S. caninervis had a range of strategies from near constitutive (adult shoots) to demonstrably inducible (protonema). A new response variable for assessing degree of DT is introduced as the minimum rate of drying from which full recovery occurs.

Exosomal miR-221 derived from hydroquinone-transformed malignant human bronchial epithelial cells is involved in cell viability of recipient cells.

miR-221, an oncogenic microRNA, can promote cell proliferation and is highly expressed in various types of tumors. However, the role of exosomal miR-221 in benzene-caused carcinogenesis remains elusive. Our study was designed to investigate whether exosomes secreted by the hydroquinone (HQ; an active metabolite of benzene)-transformed malignant cells can transmit miR-221 to normal recipient cells and its possible effects on cell viability. Our investigation revealed that expression levels of miR-221 were significantly increased in HQ-transformed malignant cells relative to normal controls. Furthermore, exposure of control cells to exosomes that were derived from HQ-transformed malignant cells increased miR-221 levels and promoted their proliferation. Analyses of the biological potency of exosomes derived from HQ-transformed malignant cells in which miR-221 levels were decreased using an inhibitor, showed that both miR-221 levels and proliferation of recipient cells were decreased, but still were higher than those of normal 16HBE cells. Our study indicates that exosomal miR-221 derived from HQ-transformed malignant human bronchial epithelial cells is involved in the proliferation of recipient cells.

NEIL1 protects against aflatoxin-induced hepatocellular carcinoma in mice.

Global distribution of hepatocellular carcinomas (HCCs) is dominated by its incidence in developing countries, accounting for >700,000 estimated deaths per year, with dietary exposures to aflatoxin (AFB1) and subsequent DNA adduct formation being a significant driver. Genetic variants that increase individual susceptibility to AFB1-induced HCCs are poorly understood. Herein, it is shown that the DNA base excision repair (BER) enzyme, DNA glycosylase NEIL1, efficiently recognizes and excises the highly mutagenic imidazole ring-opened AFB1-deoxyguanosine adduct (AFB1-Fapy-dG). Consistent with this in vitro result, newborn mice injected with AFB1 show significant increases in the levels of AFB1-Fapy-dG in Neil1-/- vs. wild-type liver DNA. Further, Neil1-/- mice are highly susceptible to AFB1-induced HCCs relative to WT controls, with both the frequency and average size of hepatocellular carcinomas being elevated in Neil1-/- The magnitude of this effect in Neil1-/- mice is greater than that previously measured in Xeroderma pigmentosum complementation group A (XPA) mice that are deficient in nucleotide excision repair (NER). Given that several human polymorphic variants of NEIL1 are catalytically inactive for their DNA glycosylase activity, these deficiencies may increase susceptibility to AFB1-associated HCCs.

Inspiratory muscle training at sea level improves the strength of inspiratory muscles during load carriage in cold-hypoxia.

Inspiratory muscle training (IMT) and functional IMT (IMTF: exercise-specific IMT activities) has been unsuccessful in reducing respiratory muscle fatigue following load carriage. IMTF did not include load carriage specific exercises. Fifteen participants split into two groups (training and control) walked 6 km loaded (18.2 kg) at speeds representing ∼50%V̇O2max in cold-hypoxia. The walk was completed at baseline; post 4 weeks IMT and 4 weeks IMTF (five exercises engaging core muscles, three involved load). The training group completed IMT and IMTF at a higher maximal inspiratory pressure (Pimax) than controls. Improvements in Pimax were greater in the training group post-IMT (20.4%, p = .025) and post-IMTF (29.1%, p = .050) compared to controls. Respiratory muscle fatigue was unchanged (p = .643). No other physiological or subjective measures were improved by IMT or IMTF. Both IMT and IMTF increased the strength of respiratory muscles pre-and-post a 6 km loaded walk in cold-hypoxia. Practitioner Summary: To explore the interaction between inspiratory muscle training (IMT), load carriage and environment, this study investigated 4 weeks IMT and 4 weeks functional IMT on respiratory muscle strength and fatigue. Functional IMT improved inspiratory muscle strength pre-and-post a loaded walk in cold-hypoxia but had no more effect than IMT alone. Abbreviations: ANOVA: analysis of variance; BF: breathing frequency; CON: control group; EELV: end-expiratory lung volume; EXP: experimental group; FEV1: forced expiratory volume in one second; FiO2: fraction of inspired oxygen; FVC: forced vital capacity; HR: heart rate; IMT: inspiratory muscle training; IMTF: functional inspiratory muscle training; Pemax: maximal expiratory pressure; Pimax: maximal inspiratory pressure; RMF: respiratory muscle fatigue; RPE: rate of perceived exertion; RWU: respiratory muscle warm-up; SaO2: arterial oxygen saturation; SpO2: peripheral oxygen saturation; V̇E: minute ventilation; V̇O2: rate of oxygen uptake.

Pilot Study of an Online Self-Directed Learning Module for Medical Decision-Making Capacity.

OBJECTIVE: This study assessed the impact of an online, self-directed learning module on medical student knowledge and attitudes regarding decision-making capacity. METHODS: The authors created a 15-min didactic video on decision-making capacity and sorted medical students on the psychiatry clerkship to teaching as usual or teaching as usual plus watching the video. The authors assessed student knowledge about and attitudes toward decisional capacity assessment using a pre/posttest design. RESULTS: Thirty-eight students completed the study (24% of all psychiatry clerkship students in one academic year). Students who watched the video (n = 14) achieved higher scores on the posttest compared with students who did not watch the video (n = 24) (90% vs 84% on general knowledge and case examples), though this difference was small and not statistically significant. The degree of improvement from pretest to posttest was higher for students who watched the video but did not reach statistical significance. CONCLUSIONS: Overall, students found the online teaching module to be a helpful augmentation strategy for learning decisional capacity assessment. The authors incorporated student feedback and additional review to create an enhanced video, which is available on ADMSEP's Clinical Simulation Initiative website.

Understanding Assessment Systems for Clinical Competency Committee Decisions: Evidence from a Multisite Study of Psychiatry Residency Training Programs.

OBJECTIVE: This multisite study examines how clinical competency committees in Psychiatry synthesize resident assessments to inform milestones decisions to provide guidelines that support their use. METHODS: The study convened training directors and associate training directors from three psychiatry residency programs to examine decision-making processes of clinical competency committees. Annual resident assessments for one second year and one third year resident were used in a mock clinical competency committee format to assign milestones for two consecutive reporting periods. The committees reflected on the process and rated how the assessment tools impacted the assessment of milestones and evaluated the overall process. The authors compared reliability of assessment between the mock committees and examined both reliability of end of rotation assessments and their composite scores when combined with clinical skills evaluations. RESULTS: End of rotation evaluations were the most informative tool for assigning milestones and clarifying discrepancies in performance. In particular, the patient care and medical knowledge competencies were the easiest to rate, while the systems-based practice and practice-based learning and improvement were the most difficult. Reliability between committees was low although higher number of available evaluations improved reliability in decision-making. CONCLUSIONS: The results indicate that the medical knowledge and patient care competencies are the easiest to rate and informed most by end of rotation evaluations and clinical skills examinations. Other evaluation tools may better capture performance on specific sub-competencies beyond workplace-based assessment, or it may be helpful to reconsider the utility of how individual sub-competencies are evaluated.

Morphological and physiological traits in relation to carbon balance in a diverse clade of dryland mosses.

Plant functional trait analyses have focused almost exclusively on vascular plants, but bryophytes comprise ancient and diverse plant lineages that have widespread global distributions and important ecological functions in terrestrial ecosystems. We examined a diverse clade of dryland mosses, Syntrichia, and studied carbon balance during a precipitation event (C-balance), a functional trait related to physiological functioning, desiccation tolerance, survival, and ecosystem carbon and nitrogen cycling. We examined variability in C-balance among 14 genotypes of Syntrichia and measured an additional 10 physiological and 13 morphological traits at the cell, leaf, shoot, and clump level. C-balance varied 20-fold among genotypes, and highest C-balances were associated with long, narrow leaves with awns, and small cells with thick cell walls, traits that may influence water uptake and retention during a precipitation event. Ordination analyses revealed that the axis most strongly correlated with C-balance included the maximum chlorophyll fluorescence, Fm , indicating the importance of photosystem II health for C exchange. C-balance represents a key functional trait in bryophytes, but its measurement is time intensive and not feasible to measure on large scales. We propose two models (using physiological and morphological traits) to predict C-balance, whereby identifying simpler to measure traits for trait databases.

Aflatoxin-Guanine DNA Adducts and Oxidatively Induced DNA Damage in Aflatoxin-Treated Mice in Vivo as Measured by Liquid Chromatography-Tandem Mass Spectrometry with Isotope Dilution.

Dietary exposure to aflatoxin B1 (AFB1) is a significant contributor to the incidence of hepatocellular carcinomas globally. AFB1 exposure leads to the formation of AFB1-N7-guanine (AFB1-N7-Gua) and two diastereomers of the imidazole ring-opened 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl-formamido)-9-hydroxyaflatoxin B1 (AFB1-FapyGua) in DNA. These adducts lead to G → T transversion mutations with the ring-opened adduct being more mutagenic than the cationic species. Accurate measurement of these three adducts as biomarkers in DNA and urine will help identify dietary exposure to AFB1 as a risk factor in the development of hepatocellular carcinoma worldwide. Herein, we report an improved methodology for the measurement of AFB1-N7-Gua and the two diastereomers of AFB1-FapyGua using liquid chromatography-tandem mass spectrometry with isotope dilution. We measured the levels of these compounds in liver DNA of six control mice and six AFB1-treated mice. Levels varying from 1.5 to 45 lesions/106 DNA bases in AFB1-treated mice were detected depending on the compound and animal. No background levels of these adducts were detected in control mice. We also tested whether the AFB1 treatment caused oxidatively induced DNA base damage using gas chromatography-tandem mass spectrometry with isotope dilution. Although background levels of several pyrimidine- and purine-derived lesions were detected, no increases in these levels were found upon AFB1 treatment of mice. On the other hand, significantly increased levels of (5' R)- and (5' S)-8,5'-cyclo-2'-deoxyadenosines were observed in liver DNA of AFB1-treated mice. The impact of this work is expected to achieve the accurate measurement of three AFB1-DNA adducts and oxidatively induced DNA lesions as biomarkers of AFB1 exposure as germane to investigations designed for the prevention of aflatoxin-related hepatocellular carcinomas and for determining the effects of genetic deficiencies in human populations.

DNA polymerase ζ limits chromosomal damage and promotes cell survival following aflatoxin exposure.

Routine dietary consumption of foods that contain aflatoxins is the second leading cause of environmental carcinogenesis worldwide. Aflatoxin-driven mutagenesis is initiated through metabolic activation of aflatoxin B1 (AFB1) to its epoxide form that reacts with N7 guanine in DNA. The resulting AFB1-N7-dG adduct undergoes either spontaneous depurination or imidazole-ring opening yielding formamidopyrimidine AFB1 (AFB1-Fapy-dG). Because this latter adduct is known to persist in human tissues and contributes to the high frequency G-to-T mutation signature associated with many hepatocellular carcinomas, we sought to establish the identity of the polymerase(s) involved in processing this lesion. Although our previous biochemical analyses demonstrated the ability of polymerase ζ (pol ζ) to incorporate an A opposite AFB1-Fapy-dG and extend from this mismatch, biological evidence supporting a unique role for this polymerase in cellular tolerance following aflatoxin exposure has not been established. Following challenge with AFB1, survival of mouse cells deficient in pol ζ (Rev3L-/-) was significantly reduced relative to Rev3L+/- cells or Rev3L-/- cells complemented through expression of the wild-type human REV3L. Furthermore, cell-cycle progression of Rev3L-/- mouse embryo fibroblasts was arrested in late S/G2 following AFB1 exposure. These Rev3L-/- cells showed an increase in replication-dependent formation of γ-H2AX foci, micronuclei, and chromosomal aberrations (chromatid breaks and radials) relative to Rev3L+/- cells. These data suggest that pol ζ is essential for processing AFB1-induced DNA adducts and that, in its absence, cells do not have an efficient backup polymerase or a repair/tolerance mechanism facilitating survival.