Mismatch discrimination and sequence bias during end-joining by DNA ligases.

DNA ligases, critical enzymes for in vivo genome maintenance and modern molecular biology, catalyze the joining of adjacent 3'-OH and 5'-phosphorylated ends in DNA. To determine whether DNA annealing equilibria or properties intrinsic to the DNA ligase enzyme impact end-joining ligation outcomes, we used a highly multiplexed, sequencing-based assay to profile mismatch discrimination and sequence bias for several ligases capable of efficient end-joining. Our data reveal a spectrum of fidelity and bias, influenced by both the strength of overhang annealing as well as sequence preferences and mismatch tolerances that vary both in degree and kind between ligases. For example, while T7 DNA ligase shows a strong preference for ligating high GC sequences, other ligases show little GC-dependent bias, with human DNA Ligase 3 showing almost none. Similarly, mismatch tolerance varies widely among ligases, and while all ligases tested were most permissive of G:T mismatches, some ligases also tolerated bulkier purine:purine mismatches. These comprehensive fidelity and bias profiles provide insight into the biology of end-joining reactions and highlight the importance of ligase choice in application design.

The relationships of sexually harassing behaviors to organizational context factors and working men's dark personality traits.

This research examined the roles of organization contexts factors and dark personality traits in men's (N = 600) self-reports of sexually harassing behaviors toward women in the workplace. Four organization context factors (a permissive climate, a masculinized job/gender context, male/female contact, and Masculinity Contest Culture [MCC] Norms) and four dark personality traits (psychopathy, narcissism, Machiavellianism, and sadism) were examined. While only one organizational context factor, MCC Norms correlated with men's admissions of sexually harassing behaviors at work, all four dark personality traits evidenced significant correlations. In a multiple regression analysis, MCC Norms emerged again as the single organizational context predictor and psychopathy as the single personality predictor of men's admissions of sexually harassing behaviors at work. Moderation analyses showed that a masculinized job/gender context interacted with psychopathy to produce more admissions of sexually harassing behaviors. Mediation analyses showed that psychopathy, narcissism, and Machiavellianism had indirect relationships with admissions of sexually harassing behaviors through MCC Norms. Higher levels on these traits were related to higher levels of these workplace norms which, in turn, predicted more admissions of sexually harassing behavior. This research sheds new light on how both organizational contexts and enduring personal characteristics of men are related to sexual harassment in the workplace.

Structural accommodation of ribonucleotide incorporation by the DNA repair enzyme polymerase Mu.

While most DNA polymerases discriminate against ribonucleotide triphosphate (rNTP) incorporation very effectively, the Family X member DNA polymerase µ (Pol µ) incorporates rNTPs almost as efficiently as deoxyribonucleotides. To gain insight into how this occurs, here we have used X-ray crystallography to describe the structures of pre- and post-catalytic complexes of Pol µ with a ribonucleotide bound at the active site. These structures reveal that Pol µ binds and incorporates a rNTP with normal active site geometry and no distortion of the DNA substrate or nucleotide. Moreover, a comparison of rNTP incorporation kinetics by wildtype and mutant Pol µ indicates that rNTP accommodation involves synergistic interactions with multiple active site residues not found in polymerases with greater discrimination. Together, the results are consistent with the hypothesis that rNTP incorporation by Pol µ is advantageous in gap-filling synthesis during DNA double strand break repair by nonhomologous end joining, particularly in nonreplicating cells containing very low deoxyribonucleotide concentrations.

Polµ tumor variants decrease the efficiency and accuracy of NHEJ.

The non homologous end-joining (NHEJ) pathway of double-strand break (DSB) repair often requires DNA synthesis to fill the gaps generated upon alignment of the broken ends, a complex task performed in human cells by two specialized DNA polymerases, Polλ and Polµ. It is now well established that Polµ is the one adapted to repair DSBs with non-complementary ends, the most challenging scenario, although the structural basis and physiological implications of this adaptation are not fully understood. Here, we demonstrate that two human Polµ point mutations, G174S and R175H, previously identified in two different tumor samples and affecting two adjacent residues, limit the efficiency of accurate NHEJ by Polµ in vitro and in vivo. Moreover, we show that this limitation is the consequence of a decreased template dependency during NHEJ, which renders the error-rate of the mutants higher due to the ability of Polµ to randomly incorporate nucleotides at DSBs. These results highlight the relevance of the 8 kDa domain of Polµ for accurate and efficient NHEJ, but also its contribution to the error-prone behavior of Polµ at 2-nt gaps. This work provides the first demonstration that mutations affecting Polµ identified in tumors can alter the efficiency and fidelity of NHEJ.

Differential item functioning for items in Berger's HIV Stigma Scale: an analysis of cohorts from the Indian, Swedish, and US contexts.

PURPOSE: To examine whether items in Berger's HIV Stigma Scale function differently with persons of different age, gender, and cultural backgrounds. METHODS: Secondary data from cohorts, collected in South India (n = 250), Sweden (n = 193), and the US (n = 603) were reanalyzed to evaluate DIF within, between, and across these cohorts. All participants had answered the revised version of the HIV stigma scale consisting of 32 items forming the subscales Personalized stigma, Disclosure concerns, Concerns about public attitudes, and Negative self-image. Differential Item Functioning (DIF) for these items was assessed using hybrid ordinal regression-IRT technique. When DIF was detected, the cumulative impact of DIF on individual subscale scores was evaluated. RESULTS: DIF was detected for 9 items within, between, or across cohorts, but the DIF was negligible in general. Detected DIF between the Swedish and Indian cohorts had a cumulative salient impact on individual scores for the subscale Disclosure Concerns; Disclosure concerns were overestimated in the Swedish cohort and both over- and underestimated in the Indian cohort. CONCLUSIONS: The items in the 32-item version of the HIV stigma scale did not seem to be particularly prone to present DIF. The DIF between the Indian and Swedish cohort for items in the subscale Disclosure Concerns could, however, result in both type I and type II errors if scores should be compared between the Indian and Swedish cohort.

Essential role for polymerase specialization in cellular nonhomologous end joining.

Nonhomologous end joining (NHEJ) repairs chromosome breaks and must remain effective in the face of extensive diversity in broken end structures. We show here that this flexibility is often reliant on the ability to direct DNA synthesis across strand breaks, and that polymerase (Pol) µ and Pol λ are the only mammalian DNA polymerases that have this activity. By systematically varying substrate in cells, we show each polymerase is uniquely proficient in different contexts. The templating nucleotide is also selected differently, with Pol µ using the unpaired base adjacent to the downstream 5' phosphate even when there are available template sites further upstream of this position; this makes Pol µ more flexible but also less accurate than Pol λ. Loss of either polymerase alone consequently has clear and distinguishable effects on the fidelity of repair, but end remodeling by cellular nucleases and the remaining polymerase helps mitigate the effects on overall repair efficiency. Accordingly, when cells are deficient in both polymerases there is synergistic impact on NHEJ efficiency, both in terms of repair of defined substrates and cellular resistance to ionizing radiation. Pol µ and Pol λ thus provide distinct solutions to a problem for DNA synthesis that is unique to this pathway and play a key role in conferring on NHEJ the flexibility required for accurate and efficient repair.

Biomarkers in Sports and Exercise: Tracking Health, Performance, and Recovery in Athletes.

Biomarker discovery and validation is a critical aim of the medical and scientific community. Research into exercise and diet-related biomarkers aims to improve health, performance, and recovery in military personnel, athletes, and lay persons. Exercise physiology research has identified individual biomarkers for assessing health, performance, and recovery during exercise training. However, there are few recommendations for biomarker panels for tracking changes in individuals participating in physical activity and exercise training programs. Our approach was to review the current literature and recommend a collection of validated biomarkers in key categories of health, performance, and recovery that could be used for this purpose. We determined that a comprehensive performance set of biomarkers should include key markers of (a) nutrition and metabolic health, (b) hydration status, (c) muscle status, (d) endurance performance, (e) injury status and risk, and (f) inflammation. Our review will help coaches, clinical sport professionals, researchers, and athletes better understand how to comprehensively monitor physiologic changes, as they design training cycles that elicit maximal improvements in performance while minimizing overtraining and injury risk.

Stigma by association and family burden among family members of people with mental illness: the mediating role of coping.

PURPOSE: When someone has a mental illness, family members may share the experience of stigma. Past research has established that family members' experiences of stigma by association predict psychological distress and lower quality-of-life. METHODS: The present study, conducted with 503 family members of people with mental illness examined the prevalence of 14 different coping strategies. Of greater importance, we examined the role of these coping strategies as mediators of the relationships between stigma by association and family burden, on the one hand, and outcomes, such as psychological distress and quality-of-life, on the other. RESULTS: The results showed that both perceived stigma by association and family burden are associated with greater psychological distress and lower quality-of-life, and that most coping strategies mediate these relationships. CONCLUSIONS: Adaptive coping strategies were related to reduced negative outcomes, while most maladaptive coping strategies were related to enhanced negative outcomes. Implications for intervention development are discussed.

Inability of myalgic encephalomyelitis/chronic fatigue syndrome patients to reproduce VO2peak indicates functional impairment.

BACKGROUND: Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a multi-system illness characterized, in part, by increased fatigue following minimal exertion, cognitive impairment, poor recovery to physical and other stressors, in addition to other symptoms. Unlike healthy subjects and other diseased populations who reproduce objective physiological measures during repeat cardiopulmonary exercise tests (CPETs), ME/CFS patients have been reported to fail to reproduce results in a second CPET performed one day after an initial CPET. If confirmed, a disparity between a first and second CPET could serve to identify individuals with ME/CFS, would be able to document their extent of disability, and could also provide a physiological basis for prescribing physical activity as well as a metric of functional impairment. METHODS: 22 subjects diagnosed with ME/CFS completed two repeat CPETs separated by 24 h. Measures of oxygen consumption (VO2), heart rate (HR), minute ventilation (Ve), workload (Work), and respiratory exchange ratio (RER) were made at maximal (peak) and ventilatory threshold (VT) intensities. Data were analyzed using ANOVA and Wilcoxon's Signed-Rank Test (for RER). RESULTS: ME/CFS patients showed significant decreases from CPET1 to CPET2 in VO2peak (13.8%), HRpeak (9 bpm), Ve peak (14.7%), and Work@peak (12.5%). Decreases in VT measures included VO2@VT (15.8%), Ve@VT (7.4%), and Work@VT (21.3%). Peak RER was high (≥1.1) and did not differ between tests, indicating maximum effort by participants during both CPETs. If data from only a single CPET test is used, a standard classification of functional impairment based on VO2peak or VO2@VT results in over-estimation of functional ability for 50% of ME/CFS participants in this study. CONCLUSION: ME/CFS participants were unable to reproduce most physiological measures at both maximal and ventilatory threshold intensities during a CPET performed 24 hours after a prior maximal exercise test. Our work confirms that repeated CPETs warrant consideration as a clinical indicator for diagnosing ME/CFS. Furthermore, if based on only one CPET, functional impairment classification will be mis-identified in many ME/CFS participants.

Coping with stigma by association and family burden among family members of people with mental illness.

In this study, we explored stigma by association, family burden, and their impact on the family members of people with mental illness. We also studied the ways in which family members coped with these phenomena. We conducted semistructured interviews with 23 immediate family members of people with mental illness. Participants reported various experiences of stigma by association and family burden. Social exclusion, being blamed, not being taken seriously, time-consuming caregiving activities, and exhaustion appeared to be the predominant forms of stigma by association and family burden experienced by the participants. The participants used problem-focused and emotion-focused coping strategies, separately or simultaneously, to cope with the negative impact of stigma by association and family burden. The results suggest that family members should have access to services to address these problems. Social, instrumental, and emotional support should be given to family members by community members and mental health professionals.

One-pot Golden Gate Assembly of an avian infectious bronchitis virus reverse genetics system.

Avian infectious bronchitis is an acute respiratory disease of poultry of particular concern for global food security. Investigation of infectious bronchitis virus (IBV), the causative agent of avian infectious bronchitis, via reverse genetics enables deeper understanding of virus biology and a rapid response to emerging variants. Classic methods of reverse genetics for IBV can be time consuming, rely on recombination for the introduction of mutations, and, depending on the system, can be subject to genome instability and unreliable success rates. In this study, we have applied data-optimized Golden Gate Assembly design to create a rapidly executable, flexible, and faithful reverse genetics system for IBV. The IBV genome was divided into 12 fragments at high-fidelity fusion site breakpoints. All fragments were synthetically produced and propagated in E. coli plasmids, amenable to standard molecular biology techniques for DNA manipulation. The assembly can be carried out in a single reaction, with the products used directly in subsequent viral rescue steps. We demonstrate the use of this system for generation of point mutants and gene replacements. This Golden Gate Assembly-based reverse genetics system will enable rapid response to emerging variants of IBV, particularly important to vaccine development for controlling spread within poultry populations.

Structure and functional analysis of the BRCT domain of translesion synthesis DNA polymerase Rev1.

Translesion synthesis (TLS) is a pathway in which specialized, low-fidelity DNA polymerases are used to overcome replication blocks caused by DNA damage. The use of this pathway often results in somatic mutations that can drive carcinogenesis. Rev1 is a TLS polymerase found in all eukaryotes that plays a pivotal role in mediating DNA damage-induced mutagenesis. It possesses a BRCA1 C-terminal (BRCT) domain that is required for its function. The rev1-1 allele encodes a mutant form of Rev1 with a G193R substitution in this domain, which reduces the level of DNA damage-induced mutagenesis. Despite its clear importance in mutagenic TLS, the role of the BRCT domain is unknown. Here, we report the X-ray crystal structure of the yeast Rev1 BRCT domain and show that substitutions in residues constituting its phosphate-binding pocket do not affect mutagenic TLS. This suggests that the role of the Rev1 BRCT domain is not to recognize phosphate groups on protein binding partners or on DNA. We also found that residue G193 is located in a conserved turn region of the BRCT domain, and our in vivo and in vitro studies suggest that the G193R substitution may disrupt Rev1 function by destabilizing the fold of the BRCT domain.

Quantitative analysis of the efficiency and mutagenic spectra of abasic lesion bypass catalyzed by human Y-family DNA polymerases.

Higher eukaryotes encode various Y-family DNA polymerases to perform global DNA lesion bypass. To provide complete mutation spectra for abasic lesion bypass, we employed short oligonucleotide sequencing assays to determine the sequences of abasic lesion bypass products synthesized by human Y-family DNA polymerases eta (hPolη), iota (hPolι) and kappa (hPolκ). The fourth human Y-family DNA polymerase, Rev1, failed to generate full-length lesion bypass products after 3 h. The results indicate that hPolι generates mutations with a frequency from 10 to 80% during each nucleotide incorporation event. In contrast, hPolη is the least error prone, generating the fewest mutations in the vicinity of the abasic lesion and inserting dAMP with a frequency of 67% opposite the abasic site. While the error frequency of hPolκ is intermediate to those of hPolη and hPolι, hPolκ has the highest potential to create frameshift mutations opposite the abasic site. Moreover, the time (t(50)(bypass)) required to bypass 50% of the abasic lesions encountered by hPolη, hPolι and hPolκ was 4.6, 112 and 1 823 s, respectively. These t(50)(bypass) values indicate that, among the enzymes, hPolη has the highest abasic lesion bypass efficiency. Together, our data suggest that hPolη is best suited to perform abasic lesion bypass in vivo.

The influence of social norms upon behavioral expressions of implicit and explicit weight-related stigma in an interactive game.

This research explored the roles of social influence and stigma-related attitudes in how people behaved toward an overweight female in an interactive computer game. Photographs were used to manipulate whether one of the players in the game was overweight or average weight. We found that both explicit and implicit anti-fat attitudes influenced interactions with an overweight player, but only when other players ostracized the overweight player, not when they included her. Under conditions of ostracism, explicit attitudes were better predictors of more controllable behaviors, while implicit attitudes were better predictors of more automatic behaviors.

Rapid 40 kb Genome Construction from 52 Parts through Data-optimized Assembly Design.

Large DNA constructs (>10 kb) are invaluable tools for genetic engineering and the development of therapeutics. However, the manufacture of these constructs is laborious, often involving multiple hierarchical rounds of preparation. To address this problem, we sought to test whether Golden Gate assembly (GGA), an in vitro DNA assembly methodology, can be utilized to construct a large DNA target from many tractable pieces in a single reaction. While GGA is routinely used to generate constructs from 5 to 10 DNA parts in one step, we found that optimization permitted the assembly of >50 DNA fragments in a single round. We applied these insights to genome construction, successfully assembling the 40 kb T7 bacteriophage genome from up to 52 parts and recovering infectious phage particles after cellular transformation. The assembly protocols and design principles described here can be applied to rapidly engineer a wide variety of large and complex assembly targets.

Variations on a theme: eukaryotic Y-family DNA polymerases.

Most classical DNA polymerases, which function in normal DNA replication and repair, are unable to synthesize DNA opposite damage in the template strand. Thus in order to replicate through sites of DNA damage, cells are equipped with a variety of nonclassical DNA polymerases. These nonclassical polymerases differ from their classical counterparts in at least two important respects. First, nonclassical polymerases are able to efficiently incorporate nucleotides opposite DNA lesions while classical polymerases are generally not. Second, nonclassical polymerases synthesize DNA with a substantially lower fidelity than do classical polymerases. Many nonclassical polymerases are members of the Y-family of DNA polymerases, and this article focuses on the mechanisms of the four eukaryotic members of this family: polymerase eta, polymerase kappa, polymerase iota, and the Rev1 protein. We discuss the mechanisms of these enzymes at the kinetic and structural levels with a particular emphasis on how they accommodate damaged DNA substrates. Work over the last decade has shown that the mechanisms of these nonclassical polymerases are fascinating variations of the mechanism of the classical polymerases. The mechanisms of polymerases eta and kappa represent rather minor variations, while the mechanisms of polymerase iota and the Rev1 protein represent rather major variations. These minor and major variations all accomplish the same goal: they allow the nonclassical polymerases to circumvent the problems posed by the template DNA lesion.

Analysis of the outcome of intracytoplasmic sperm injection using fresh or frozen sperm.

OBJECTIVES: • To compare the outcome of first-attempt intracytoplasmic sperm injection (ICSI) ICSI--embryo transfer (ET) cycles using frozen-thawed testicular sperm (FTTS), fresh testicular sperm (FTS), frozen-thawed epididymal sperm (FTES) and fresh epididymal sperm (FES) so as to determine which of these has the most successful ICSI outcome with respect to fertilization rate (FR), pregnancy rate (PR) and birth rate. • To assess the outcomes according to the underlying aetiology of azoospermia. PATIENTS AND METHODS: • The records of 493 patients undergoing first-attempt ICSI between 1993 and 2008 were reviewed retrospectively. FTS was used in 112 cycles, FTTS in 43 cycles, FES in 279 cycles, and FTES in 59 cycles. • Within each group, the aetiology of the azoospermia was recorded according to history, clinical examination and histological analysis (n = 316). • The FR, clinical PR and delivery rate were calculated for each group with respect to the type of sperm retrieval used. RESULTS: • Analysis of the data showed no significant differences between any of the four groups in the FR, PR or delivery rate (P > 0.05). • There were no significant differences seen between fresh sperm (FTS and FES) and frozen sperm (FTTS and FTES) or between epididymal sperm (FES and FTES) and testicular sperm (FTS and FTTS) in any of the outcomes measured (P > 0.05). However, subset analysis showed a statistically higher FR and PR for FTTS over fresh sperm. • When comparing aetiologies, there was no significant difference in the FR, clinical PR and delivery rate between obstructive azoospermia (OA) and non-obstructive azoospermia (NOA) groups. However, sub-set analysis showed a higher PR and birth rate for FTTS over fresh sperm in both OA and NOA groups. CONCLUSIONS: • The results of the present study suggest that using frozen sperm in ICSI cycles is a reliable and favourable method with the same outcome as fresh sperm. • Testicular and epididymal sperm have similar ICSI outcomes for both fresh and frozen samples. However, results suggest a tendency for higher PRs and birth rates for frozen than for fresh testicular sperm in both OA and NOA aetiologies. • The aetiology of azoospermia does not significantly affect the outcome of first-attempt ICSI. The higher rates in the frozen groups suggest that these patients have had better quality semen when they were initially harvested and frozen.

HIV status disclosure among HIV-positive African and Afro-Caribbean people in the Netherlands.

HIV status disclosure is often characterized as a dilemma. On the one hand, disclosure can promote health, social support, and psychological well-being. On the other, disclosure can lead to stigmatization, rejection, and other negative social interactions. Previous research has shown that HIV status disclosure is a reasoned process whereby the costs and benefits to oneself and to others are weighed. As such, understanding disclosure requires understanding the reasons for and against disclosure employed by people living with HIV (PLWH). In this study, disclosure among a population disproportionately affected by HIV in the Netherlands, namely African and Afro-Caribbean diaspora, was investigated. Reasons for nondisclosure were fear of stigmatization, previous negative experiences with disclosure, having observed the stigmatization of other PLWH, shame, the desire to protect others - particularly one's children and family - from stigmatization by association and/or worrying, and the belief that one's HIV status is a private matter. Participants reported disclosing because they were in a close and supportive relationship, disclosure led to emotional release, disclosure could lead to emotional or financial support, they felt a perceived duty to inform, and they had a desire to educate others about sexual risk-taking. The findings suggest that stigma plays an important role in disclosure decisions among these populations. They further point to a need for HIV-related stigma reduction interventions in African and Afro-Caribbean communities and culturally sensitive counseling for PLWH whereby caregivers do not automatically assume that disclosure is best but rather provide a safe environment in which the costs and benefits of disclosure can be weighed and strategies for disclosure can be developed, if perceived as beneficial by PLWH.

Unreliability and error in the military's "gold standard" measure of sexual harassment by education and gender.

The Department of Defense's "gold standard" sexual harassment measure, the Sexual Harassment Core Measure (SHCore), is based on an earlier measure that was developed primarily in college women. Furthermore, the SHCore requires a reading grade level of 9.1. This may be higher than some troops' reading abilities and could generate unreliable estimates of their sexual harassment experiences. Results from 108 male and 96 female soldiers showed that the SHCore's temporal stability and alternate-forms reliability was significantly worse (a) in soldiers without college experience compared to soldiers with college experience and (b) in men compared to women. For men without college experience, almost 80% of the temporal variance in SHCore scores was attributable to error. A plain language version of the SHCore had mixed effects on temporal stability depending on education and gender. The SHCore may be particularly ill suited for evaluating population trends of sexual harassment in military men without college experience.

Triple-lumen peripherally inserted central catheter in patients in the critical care unit: prospective evaluation.

PURPOSE: To prospectively evaluate outcomes associated with use of a triple-lumen (TL) peripherally inserted central catheter (PICC) in the intensive care unit (ICU) setting. MATERIALS AND METHODS: Patients were prospectively enrolled in this HIPAA-compliant, institutional review board-approved study. Informed consent was obtained. All patients were in one hospital's ICUs and needed intermediate-term central venous access requiring three lumina. A 6-F tapered TL PICC was placed by a bedside nursing-based team with backup from the Interventional Radiology department. Placement complications, as well as long-term complications, were recorded. At catheter removal, ultrasonography (US) of the veins containing the TL PICC was performed to detect occult venous thrombosis. Regardless of indication for removal, catheters were sent for culture to detect colonization. RESULTS: The study was stopped prematurely after 50 of a planned 167 patients were enrolled when a scheduled interim analysis detected a venous thrombosis rate that was considered unacceptably high by the study oversight committee (thrombosis was symptomatic in 20% of patients [10 of 50]). Venous thrombosis (symptomatic or asymptomatic) was detected in 26 of 45 patients (58%; 95% confidence interval [CI]: 43%, 72%) examined with US. Documented catheter-related bloodstream infection did not occur (0%; 95% CI: 0%, 7%); colonization was detected in three of 29 catheter tips sent for culture (10%; 95% CI: 2%, 27%). Catheter malfunction and dislodgment occurred in one patient each. CONCLUSION: The TL PICC design used in this study resulted in unacceptably high venous thrombosis rates. Even when used in a high-risk setting for infection (ie, the ICU), rates of clinically evident infection and colonization were absent and low, respectively.