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.

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.

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.

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.

Enabling one-pot Golden Gate assemblies of unprecedented complexity using data-optimized assembly design.

DNA assembly is an integral part of modern synthetic biology, as intricate genetic engineering projects require robust molecular cloning workflows. Golden Gate assembly is a frequently employed DNA assembly methodology that utilizes a Type IIS restriction enzyme and a DNA ligase to generate recombinant DNA constructs from smaller DNA fragments. However, the utility of this methodology has been limited by a lack of resources to guide experimental design. For example, selection of the DNA sequences at fusion sites between fragments is based on broad assembly guidelines or pre-vetted sets of junctions, rather than being customized for a particular application or cloning project. To facilitate the design of robust assembly reactions, we developed a high-throughput DNA sequencing assay to examine reaction outcomes of Golden Gate assembly with T4 DNA ligase and the most commonly used Type IIS restriction enzymes that generate three-base and four-base overhangs. Next, we incorporated these findings into a suite of webtools that design assembly reactions using the experimental data. These webtools can be used to create customized assemblies from a target DNA sequence or a desired number of fragments. Lastly, we demonstrate how using these tools expands the limits of current assembly systems by carrying out one-pot assemblies of up to 35 DNA fragments. Full implementation of the tools developed here enables direct expansion of existing assembly standards for modular cloning systems (e.g. MoClo) as well as the formation of robust new high-fidelity standards.

Structural snapshots of human DNA polymerase µ engaged on a DNA double-strand break.

Genomic integrity is threatened by cytotoxic DNA double-strand breaks (DSBs), which must be resolved efficiently to prevent sequence loss, chromosomal rearrangements/translocations, or cell death. Polymerase µ (Polµ) participates in DSB repair via the nonhomologous end-joining (NHEJ) pathway, by filling small sequence gaps in broken ends to create substrates ultimately ligatable by DNA Ligase IV. Here we present structures of human Polµ engaging a DSB substrate. Synapsis is mediated solely by Polµ, facilitated by single-nucleotide homology at the break site, wherein both ends of the discontinuous template strand are stabilized by a hydrogen bonding network. The active site in the quaternary Pol µ complex is poised for catalysis and nucleotide incoporation proceeds in crystallo. These structures demonstrate that Polµ may address complementary DSB substrates during NHEJ in a manner indistinguishable from single-strand breaks.

Comprehensive Profiling of Four Base Overhang Ligation Fidelity by T4 DNA Ligase and Application to DNA Assembly.

Synthetic biology relies on the manufacture of large and complex DNA constructs from libraries of genetic parts. Golden Gate and other Type IIS restriction enzyme-dependent DNA assembly methods enable rapid construction of genes and operons through one-pot, multifragment assembly, with the ordering of parts determined by the ligation of Watson-Crick base-paired overhangs. However, ligation of mismatched overhangs leads to erroneous assembly, and low-efficiency Watson Crick pairings can lead to truncated assemblies. Using sets of empirically vetted, high-accuracy junction pairs avoids this issue but limits the number of parts that can be joined in a single reaction. Here, we report the use of comprehensive end-joining ligation fidelity and bias data to predict high accuracy junction sets for Golden Gate assembly. The ligation profile accurately predicted junction fidelity in ten-fragment Golden Gate assembly reactions and enabled accurate and efficient assembly of a lac cassette from up to 24-fragments in a single reaction.

Ribonucleotide incorporation enables repair of chromosome breaks by nonhomologous end joining.

The nonhomologous end-joining (NHEJ) pathway preserves genome stability by ligating the ends of broken chromosomes together. It employs end-processing enzymes, including polymerases, to prepare ends for ligation. We show that two such polymerases incorporate primarily ribonucleotides during NHEJ-an exception to the central dogma of molecular biology-both during repair of chromosome breaks made by Cas9 and during V(D)J recombination. Moreover, additions of ribonucleotides but not deoxynucleotides effectively promote ligation. Repair kinetics suggest that ribonucleotide-dependent first-strand ligation is followed by complementary strand repair with deoxynucleotides, then by replacement of ribonucleotides embedded in the first strand with deoxynucleotides. Our results indicate that as much as 65% of cellular NHEJ products have transiently embedded ribonucleotides, which promote flexibility in repair at the cost of more fragile intermediates.

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.

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.

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.

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.

Regulation of human polλ by ATM-mediated phosphorylation during non-homologous end joining.

DNA double strand breaks (DSBs) trigger a variety of cellular signaling processes, collectively termed the DNA-damage response (DDR), that are primarily regulated by protein kinase ataxia-telangiectasia mutated (ATM). Among DDR activated processes, the repair of DSBs by non-homologous end joining (NHEJ) is essential. The proper coordination of NHEJ factors is mainly achieved through phosphorylation by an ATM-related kinase, the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), although the molecular basis for this regulation has yet to be fully elucidated. In this study we identify the major NHEJ DNA polymerase, DNA polymerase lambda (Polλ), as a target for both ATM and DNA-PKcs in human cells. We show that Polλ is efficiently phosphorylated by DNA-PKcs in vitro and predominantly by ATM after DSB induction with ionizing radiation (IR) in vivo. We identify threonine 204 (T204) as a main target for ATM/DNA-PKcs phosphorylation on human Polλ, and establish that its phosphorylation may facilitate the repair of a subset of IR-induced DSBs and the efficient Polλ-mediated gap-filling during NHEJ. Molecular evidence suggests that Polλ phosphorylation might favor Polλ interaction with the DNA-PK complex at DSBs. Altogether, our work provides the first demonstration of how Polλ is regulated by phosphorylation to connect with the NHEJ core machinery during DSB repair in human cells.

Peak Running Intensity of International Rugby: Implications for Training Prescription.

PURPOSE: To quantify the duration and position-specific peak running intensities of international rugby union for the prescription and monitoring of specific training methodologies. METHODS: Global positioning systems (GPS) were used to assess the activity profile of 67 elite-level rugby union players from 2 nations across 33 international matches. A moving-average approach was used to identify the peak relative distance (m/min), average acceleration/deceleration (AveAcc; m/s2), and average metabolic power (Pmet) for a range of durations (1-10 min). Differences between positions and durations were described using a magnitude-based network. RESULTS: Peak running intensity increased as the length of the moving average decreased. There were likely small to moderate increases in relative distance and AveAcc for outside backs, halfbacks, and loose forwards compared with the tight 5 group across all moving-average durations (effect size [ES] = 0.27-1.00). Pmet demands were at least likely greater for outside backs and halfbacks than for the tight 5 (ES = 0.86-0.99). Halfbacks demonstrated the greatest relative distance and Pmet outputs but were similar to outside backs and loose forwards in AveAcc demands. CONCLUSIONS: The current study has presented a framework to describe the peak running intensities achieved during international rugby competition by position, which are considerably higher than previously reported whole-period averages. These data provide further knowledge of the peak activity profiles of international rugby competition, and this information can be used to assist coaches and practitioners in adequately preparing athletes for the most demanding periods of play.

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.

A Geographic Simulation Model for the Treatment of Trauma Patients in Disasters.

BACKGROUND: Though the US civilian trauma care system plays a critical role in disaster response, there is currently no systems-based strategy that enables hospital emergency management and local and regional emergency planners to quantify, and potentially prepare for, surges in trauma care demand that accompany mass-casualty disasters. OBJECTIVE: A proof-of-concept model that estimates the geographic distributions of patients, trauma center resource usage, and mortality rates for varying disaster sizes, in and around the 25 largest US cities, is presented. The model was designed to be scalable, and its inputs can be modified depending on the planning assumptions of different locales and for different types of mass-casualty events. METHODS: To demonstrate the model's potential application to real-life planning scenarios, sample disaster responses for 25 major US cities were investigated using a hybrid of geographic information systems and dynamic simulation-optimization. In each city, a simulated, fast-onset disaster epicenter, such as might occur with a bombing, was located randomly within one mile of its population center. Patients then were assigned and transported, in simulation, via the new model to Level 1, 2, and 3 trauma centers, in and around each city, over a 48-hour period for disaster scenario sizes of 100, 500, 5000, and 10,000 casualties. RESULTS: Across all 25 cities, total mean mortality rates ranged from 26.3% in the smallest disaster scenario to 41.9% in the largest. Out-of-hospital mortality rates increased (from 21.3% to 38.5%) while in-hospital mortality rates decreased (from 5.0% to 3.4%) as disaster scenario sizes increased. The mean number of trauma centers involved ranged from 3.0 in the smallest disaster scenario to 63.4 in the largest. Cities that were less geographically isolated with more concentrated trauma centers in their surrounding regions had lower total and out-of-hospital mortality rates. The nine US cities listed as being the most likely targets of terrorist attacks involved, on average, more trauma centers and had lower mortality rates compared with the remaining 16 cities. CONCLUSIONS: The disaster response simulation model discussed here may offer insights to emergency planners and health systems in more realistically planning for mass-casualty events. Longer wait and transport times needed to distribute high numbers of patients to distant trauma centers in fast-onset disasters may create predictable increases in mortality and trauma center resource consumption. The results of the modeled scenarios indicate the need for a systems-based approach to trauma care management during disasters, since the local trauma center network was often too small to provide adequate care for the projected patient surge. Simulation of out-of-hospital resources that might be called upon during disasters, as well as guidance in the appropriate execution of mutual aid agreements and prevention of over-response, could be of value to preparedness planners and emergency response leaders. Study assumptions and limitations are discussed. Carr BG , Walsh L , Williams JC , Pryor JP , Branas CC . A geographic simulation model for the treatment of trauma patients in disasters. Prehosp Disaster Med. 2016;31(4):413-421.

The Role of Burden and Deviation in Ostracizing Others.

Ostracism (being excluded and ignored) is a painful experience, so why do individuals ostracize others? Previous research suggests individuals often ostracize those who are deviate, but not always. We posit that there may be two types of deviation, burdensome and non-burdensome, and the former is most likely to be ostracized. Study 1 manipulated burdensome deviation by programming a group member to perform more slowly (8 or 16 sec.) than others (4 sec.) in a virtual ball-toss game. Participants perceived slower players as more burdensome and deviate than normal speed players. Additionally, participants ostracized (gave fewer ball tosses to) the slowest player. Study 2 examined participant responses to both burdensome deviation (8- and 16-sec. players) and non-burdensome deviation (goth appearance). Participants again perceived the slower players to be burdensome and deviate, and ostracized them. They perceived the goth player to be deviate but not burdensome and did not ostracize this player.

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.

Stigma as a Barrier to HIV-Related Activities Among African-American Churches in South Carolina.

South Carolina has one of the highest HIV/AIDS prevalence rates in the United States. More than 70% of those infected are African American. Traditionally, Black churches have been one of the primary sources of health outreach programs in Southern African-American communities. In this research, we explored the role of HIV-related stigma as a barrier to the acceptance of HIV-related activities in Black churches. A survey of African-American adults in South Carolina found that the overall level of stigma associated with HIV/AIDS was comparable to what has been found in a national probability sample of people in the United States. Consistent with the stigma-as-barrier hypothesis, the degree to which survey respondents endorsed HIV-related stigma was related to less positive attitudes concerning the involvement of Black churches in HIV-related activities.

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.