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.

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.

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 DNA assembly methodology, can be utilized to construct a large DNA target from many tractable pieces in a single reaction.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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/s), and average metabolic power (P) for a range of durations (1-10 min).

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.

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.

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.

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.

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.

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.

The fidelity of the ligation step determines how ends are resolved during nonhomologous end joining.

Nonhomologous end joining (NHEJ) can effectively resolve chromosome breaks despite diverse end structures; however, it is unclear how the steps employed for resolution are determined. We sought to address this question by analysing cellular NHEJ of ends with systematically mispaired and damaged termini. We show NHEJ is uniquely proficient at bypassing subtle terminal mispairs and radiomimetic damage by direct ligation.

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.

Nonhomologous end joining: a good solution for bad ends.

Double strand breaks pose unique problems for DNA repair, especially when broken ends possess complex structures that interfere with standard DNA transactions. Nonhomologous end joining can use multiple strategies to solve these problems. It further uses sophisticated means to ensure the strategy chosen provides the ideal balance of flexibility and accuracy.

Sustained active site rigidity during synthesis by human DNA polymerase μ.

DNA polymerase μ (Pol μ) is the only template-dependent human DNA polymerase capable of repairing double-strand DNA breaks (DSBs) with unpaired 3' ends in nonhomologous end joining (NHEJ). To probe this function, we structurally characterized Pol μ's catalytic cycle for single-nucleotide incorporation. These structures indicate that, unlike other template-dependent DNA polymerases, Pol μ shows no large-scale conformational changes in protein subdomains, amino acid side chains or DNA upon dNTP binding or catalysis.

Peer bystanders to bullying: who wants to play with the victim?

Given widespread concern associated with school-based bullying, researchers have looked beyond a dyadic perspective (i.e., bullies and victims only), and now consider the broader social ecology of the peer group.