Mechanics and Single-Molecule Interrogation of DNA Recombination.

The repair of DNA by homologous recombination is an essential, efficient, and high-fidelity process that mends DNA lesions formed during cellular metabolism; these lesions include double-stranded DNA breaks, daughter-strand gaps, and DNA cross-links. Genetic defects in the homologous recombination pathway undermine genomic integrity and cause the accumulation of gross chromosomal abnormalities-including rearrangements, deletions, and aneuploidy-that contribute to cancer formation. Recombination proceeds through the formation of joint DNA molecules-homologously paired but metastable DNA intermediates that are processed by several alternative subpathways-making recombination a versatile and robust mechanism to repair damaged chromosomes. Modern biophysical methods make it possible to visualize, probe, and manipulate the individual molecules participating in the intermediate steps of recombination, revealing new details about the mechanics of genetic recombination. We review and discuss the individual stages of homologous recombination, focusing on common pathways in bacteria, yeast, and humans, and place particular emphasis on the molecular mechanisms illuminated by single-molecule methods.

BRCA2 chaperones RAD51 to single molecules of RPA-coated ssDNA.

Mutations in the breast cancer susceptibility gene, BRCA2, greatly increase an individual's lifetime risk of developing breast and ovarian cancers. BRCA2 suppresses tumor formation by potentiating DNA repair via homologous recombination. Central to recombination is the assembly of a RAD51 nucleoprotein filament, which forms on single-stranded DNA (ssDNA) generated at or near the site of chromosomal damage. However, replication protein-A (RPA) rapidly binds to and continuously sequesters this ssDNA, imposing a kinetic barrier to RAD51 filament assembly that suppresses unregulated recombination. Recombination mediator proteins-of which BRCA2 is the defining member in humans-alleviate this kinetic barrier to catalyze RAD51 filament formation. We combined microfluidics, microscopy, and micromanipulation to directly measure both the binding of full-length BRCA2 to-and the assembly of RAD51 filaments on-a region of RPA-coated ssDNA within individual DNA molecules designed to mimic a resected DNA lesion common in replication-coupled recombinational repair. We demonstrate that a dimer of RAD51 is minimally required for spontaneous nucleation; however, growth self-terminates below the diffraction limit. BRCA2 accelerates nucleation of RAD51 to a rate that approaches the rapid association of RAD51 to naked ssDNA, thereby overcoming the kinetic block imposed by RPA. Furthermore, BRCA2 eliminates the need for the rate-limiting nucleation of RAD51 by chaperoning a short preassembled RAD51 filament onto the ssDNA complexed with RPA. Therefore, BRCA2 regulates recombination by initiating RAD51 filament formation.

Assembly mechanism and cryoEM structure of RecA recombination nucleofilaments from Streptococcus pneumoniae.

RecA-mediated homologous recombination (HR) is a key mechanism for genome maintenance and plasticity in bacteria. It proceeds through RecA assembly into a dynamic filament on ssDNA, the presynaptic filament, which mediates DNA homology search and ordered DNA strand exchange. Here, we combined structural, single molecule and biochemical approaches to characterize the ATP-dependent assembly mechanism of the presynaptic filament of RecA from Streptococcus pneumoniae (SpRecA), in comparison to the Escherichia coli RecA (EcRecA) paradigm. EcRecA polymerization on ssDNA is assisted by the Single-Stranded DNA Binding (SSB) protein, which unwinds ssDNA secondary structures that block EcRecA nucleofilament growth. We report by direct microscopic analysis of SpRecA filamentation on ssDNA that neither of the two paralogous pneumococcal SSBs could assist the extension of SpRecA nucleopolymers. Instead, we found that the conserved RadA helicase promotes SpRecA nucleofilamentation in an ATP-dependent manner. This allowed us to solve the atomic structure of such a long native SpRecA nucleopolymer by cryoEM stabilized with ATPγS. It was found to be equivalent to the crystal structure of the EcRecA filament with a marked difference in how RecA mediates nucleotide orientation in the stretched ssDNA. Then, our results show that SpRecA and EcRecA HR activities are different, in correlation with their distinct ATP-dependent ssDNA binding modes.

Post-Concussion Symptoms, Cognition and Brain Connectivity in an Australian Undergraduate Population: A Quantitative Electroencephalography Study.

BACKGROUND: An estimated 99 in 100,000 people experience a traumatic brain injury (TBI), with 85% being mild (mTBI) in nature. The Post-Concussion Symptom Scale (PCSS), is a reliable and valid measure of post-mTBI symptoms; however, diagnostic specificity is challenging due to high symptom rates in the general population. Understanding the neurobiological characteristics that distinguish high and low PCSS raters may provide further clarification on this phenomenon. AIM: To explore the neurobiological characteristics of post-concussion symptoms through the association between PCSS scores, brain network connectivity (using quantitative electroencephalography; qEEG) and cognition in undergraduates. HYPOTHESES: high PCSS scorers will have (1) more network dysregulation and (2) more cognitive dysfunction compared to the low PCSS scorers. METHODS: A sample of 40 undergraduates were divided into high and low PCSS scorers. Brain connectivity was measured using qEEG, and cognition was measured via neuropsychological measures of sustained attention, inhibition, immediate attention, working memory, processing speed and inhibition/switching. RESULTS: Contrary to expectations, greater frontoparietal network dysregulation was seen in the low PCSS score group (p = 0.003). No significant difference in cognitive dysfunction was detected between high and low PCSS scorers. Post-hoc analysis in participants who had experienced mTBI revealed greater network dysregulation in those reporting a more recent mTBI. CONCLUSIONS: Measuring post-concussion symptoms alone is not necessarily informative about changes in underlying neural mechanisms. In an exploratory subset analysis, brain network dysregulation appears to be greater in the early post-injury phase compared to later. Further analysis of underlying PCSS constructs and how to measure these in a non-athlete population and clinical samples is warranted.

Contribution of higher-order structure to perception of mirror symmetry: Role of shapes and corners.

Visual mirror symmetry is a global feature that is dependent on specific low-level relationships between component elements. Initially conceptualized as virtual lines between paired elements, it has been suggested that higher-order structure between pairs of symmetric elements forming virtual four cornered shapes may also be important for strengthening the percept of mirror symmetry. We utilize corner elements, formed by joining two Gabor elements along a central midline creating vertices with variable internal angles, in a temporal integration paradigm. This allows us to specifically manipulate the presence and type of higher-order versus lower-order structure in patterns with symmetrically placed elements. We show a significant contribution of higher-order structure to the salience of visual symmetries compared with patterns with only lower-order structures. We also find that although we are more sensitive to patterns with higher-order structure, there is no difference in the temporal processing of higher-order versus lower-order patterns. These findings have important implications for existing spatial filter models of symmetry perception that rely on lower-order structures alone and reinforces the need for elaborated models that can more readily capture the complexities of real-world symmetries.

The Impact of Transparency and Decision Risk on Human-Automation Teaming Outcomes.

OBJECTIVE: Examine the effects of decision risk and automation transparency on the accuracy and timeliness of operator decisions, automation verification rates, and subjective workload. BACKGROUND: Decision aids typically benefit performance, but can provide incorrect advice due to contextual factors, creating the potential for automation disuse or misuse. Decision aids can reduce an operator's manual problem evaluation, and it can also be strategic for operators to minimize verifying automated advice in order to manage workload. METHOD: Participants assigned the optimal unmanned vehicle to complete missions. A decision aid provided advice but was not always reliable. Two levels of decision aid transparency were manipulated between participants. The risk associated with each decision was manipulated using a financial incentive scheme. Participants could use a calculator to verify automated advice; however, this resulted in a financial penalty. RESULTS: For high- compared with low-risk decisions, participants were more likely to reject incorrect automated advice and were more likely to verify automation and reported higher workload. Increased transparency did not lead to more accurate decisions and did not impact workload but decreased automation verification and eliminated the increased decision time associated with high decision risk. CONCLUSION: Increased automation transparency was beneficial in that it decreased automation verification and decreased decision time. The increased workload and automation verification for high-risk missions is not necessarily problematic given the improved automation correct rejection rate. APPLICATION: The findings have potential application to the design of interfaces to improve human-automation teaming, and for anticipating the impact of decision risk on operator behavior.

Trustworthiness perception is mandatory: Task instructions do not modulate fast periodic visual stimulation trustworthiness responses.

Although it is often assumed that humans spontaneously respond to the trustworthiness of others' faces, it is still unclear whether responses to facial trust are mandatory or can be modulated by instructions. Considerable scientific interest lies in understanding whether trust processing is mandatory, given the societal consequences of biased trusting behavior. We tested whether neural responses indexing trustworthiness discrimination depended on whether the task involved focusing on facial trustworthiness or not, using a fast periodic visual stimulation electroencephalography oddball paradigm with a neural marker of trustworthiness discrimination at 1 Hz. Participants judged faces on size without any reference to trust, explicitly formed impressions of facial trust, or were given a financial lending context that primed trust, without explicit trust judgement instructions. Significant trustworthiness discrimination responses at 1 Hz were found in all three conditions, demonstrating the robust nature of trustworthiness discrimination at the neural level. Moreover, no effect of task instruction was observed, with Bayesian analyses providing moderate to decisive evidence that task instruction did not affect trustworthiness discrimination. Our finding that visual trustworthiness discrimination is mandatory points to the remarkable spontaneity of trustworthiness processing, providing clues regarding why these often unreliable impressions are ubiquitous.

A Pan-RNase Inhibitor Enabling CRISPR-mRNA Platforms for Engineering of Primary Human Monocytes.

Monocytes and their downstream effectors are critical components of the innate immune system. Monocytes are equipped with chemokine receptors, allowing them to migrate to various tissues, where they can differentiate into macrophage and dendritic cell subsets and participate in tissue homeostasis, infection, autoimmune disease, and cancer. Enabling genome engineering in monocytes and their effector cells will facilitate a myriad of applications for basic and translational research. Here, we demonstrate that CRISPR-Cas9 RNPs can be used for efficient gene knockout in primary human monocytes. In addition, we demonstrate that intracellular RNases are likely responsible for poor and heterogenous mRNA expression as incorporation of pan-RNase inhibitor allows efficient genome engineering following mRNA-based delivery of Cas9 and base editor enzymes. Moreover, we demonstrate that CRISPR-Cas9 combined with an rAAV vector DNA donor template mediates site-specific insertion and expression of a transgene in primary human monocytes. Finally, we demonstrate that SIRPa knock-out monocyte-derived macrophages have enhanced activity against cancer cells, highlighting the potential for application in cellular immunotherapies.

Sequential assembly of the septal cell envelope prior to V snapping in Corynebacterium glutamicum.

Members of the Corynebacterineae, including Corynebacterium and Mycobacterium, have an atypical cell envelope characterized by an additional mycomembrane outside of the peptidoglycan layer. How this multilayered cell envelope is assembled remains unclear. Here, we tracked the assembly dynamics of different envelope layers in Corynebacterium glutamicum and Mycobacterium smegmatis by using metabolic labeling and found that the septal cell envelope is assembled sequentially in both species. Additionally, we demonstrate that in C. glutamicum, the peripheral peptidoglycan layer at the septal junction remains contiguous throughout septation, forming a diffusion barrier for the fluid mycomembrane. This diffusion barrier is resolved through perforations in the peripheral peptidoglycan, thus leading to the confluency of the mycomembrane before daughter cell separation (V snapping). Furthermore, the same junctional peptidoglycan also serves as a mechanical link holding the daughter cells together and undergoes mechanical fracture during V snapping. Finally, we show that normal V snapping in C. glutamicum depends on complete assembly of the septal cell envelope.

RecA: Regulation and Mechanism of a Molecular Search Engine.

Homologous recombination maintains genomic integrity by repairing broken chromosomes. The broken chromosome is partially resected to produce single-stranded DNA (ssDNA) that is used to search for homologous double-stranded DNA (dsDNA). This homology driven 'search and rescue' is catalyzed by a class of DNA strand exchange proteins that are defined in relation to Escherichia coli RecA, which forms a filament on ssDNA. Here, we review the regulation of RecA filament assembly and the mechanism by which RecA quickly and efficiently searches for and identifies a unique homologous sequence among a vast excess of heterologous DNA. Given that RecA is the prototypic DNA strand exchange protein, its behavior affords insight into the actions of eukaryotic RAD51 orthologs and their regulators, BRCA2 and other tumor suppressors.

Case report of amniotic fluid embolism coagulopathy following abortion; use of viscoelastic point-of-care analysis.

BACKGROUND: Amniotic fluid embolism (AFE) is a rare, life threatening obstetric complication, often associated with severe coagulopathy. Induced abortions are extremely safe procedures however complications including AFE can occur. CASE PRESENTATION: A 29-year-old previously healthy woman, gravida 1 para 0, presented for a scheduled second trimester induced abortion via dilation and evacuation at 22-weeks gestation. The case was complicated by a suspected AFE with associated profound coagulopathy. Viscoelastic point-of-care coagulation analysis was used to successfully and swiftly guide management of her coagulopathy. CONCLUSION: AFE can occur in the setting of induced abortion. This case report suggests viscoelastic point-of-care coagulation analyzers may aid in the management of pregnancy-related coagulopathy by providing faster coagulation assessment than laboratory testing, and facilitating timely, targeted management of coagulopathy.

The effectiveness of touchscreen-based attentional bias modification to thin body stimuli on state rumination.

Ruminative thinking is considered a vulnerability factor for eating disorder symptomatology. Research suggests that attentional bias to body shape stimuli may serve to underpin this maladaptive form of emotion regulation. The current study aimed to determine the direct effect of attentional bias to thin-ideal bodies on state depressive rumination. Additionally, this study sought to evaluate the efficacy of attentional bias modification (ABM) utilising a touchscreen device. A well-established ABM protocol, the modified dot probe task, was used for both attentional assessment and training. Female undergraduate students (N = 110) completed an ABM session where attention was trained either towards, or away from, thin-ideal body images. Pre- and post-attentional training, participants completed the dot probe task, as well as a state measure of depressive rumination. Results revealed that the ABM training induced a greater attentional bias to thin-ideal bodies in the attend-thin training condition than in the avoid-thin training condition. Furthermore, induced attentional avoidance of thin-ideal bodies led to a significant reduction in state depressive rumination. The current findings suggest that touchscreen-based ABM is effective in modifying patterns of attentional bias and state depressive rumination.

Can People Accurately Estimate the Calories in Food Images? An Optimised Set of Low- and High- Calorie Images from the food-pics database.

Calorie intake plays an important role in maintaining a healthy weight. As such, researchers often use the calorie content of food as a distinction when investigating appetite related brain processes and eating behaviour. This distinction assumes that observers accurately perceive caloric content. However, there is evidence suggesting this is not always the case. The current study examined how accurately observers could estimate the caloric content of food images from the widely used "Food-pics" database. Eight hundred and forty psychology undergraduate students (aged 16-60, 64% female) estimated the caloric value of 178 high and 182 low calorie foods. Calorie content of food from both categories was significantly overestimated. Additionally, 7.7% of low calorie images were misperceived as being high calorie images and 35% of high calorie images were misperceived as being low calorie foods. Neither participants' gender, nor the recognisability and likability of the food images, influenced calorie estimation. Our findings show that most people are unable to accurately estimate caloric content of most food. Despite this, a selection of food images were judged accurately, and we advocate the use of these in research where it is important to have low- and high-calorie food images. Specifically, we propose an optimised stimulus set of 25 high and 25 low calorie food images that are accurately judged by adult participants. In addition, we provide the open source dataset of our ratings of Food-pics images which, when added to the existing Food-pics attributes, creates an enhanced tool for researchers selecting food stimuli.

Evidence for a perceptual mechanism relating body size misperception and eating disorder symptoms.

PURPOSE: There are known and serious health risks associated with extreme body weights, including the development of eating disorders. Body size misperceptions are particularly evident in individuals with eating disorders, compared to healthy controls. The present research investigated whether serial dependence, a recently discovered bias in body size judgement, is associated with eating disorder symptomatology. We additionally examined whether this bias operates on holistic body representations or whether it works by distorting specific visual features. METHODS: A correlational analysis was used to examine the association between serial dependence and eating disorder symptomatology. We used a within-subjects experimental design to investigate the holistic nature of this misperception. Participants were 63 young women, who judged the size of upright and inverted female body images using a visual analogue scale and then completed the Eating Disorder Examination-Questionnaire (EDE-Q) to assess eating disorder symptoms. RESULTS: Our findings provide the first evidence of an association between serial dependence and eating disorder symptoms, with significant and positive correlations between body size misperception owing to serial dependence and EDE-Q scores, when controlling for Body Mass Index. Furthermore, we reveal that serial dependence is consistent with distortion of local visual features. CONCLUSIONS: Findings are discussed in relation to the broader theories of central coherence, cognitive inflexibility, and multisensory integration difficulties, and as providing a candidate mechanism for body size misperception in an eating disorder population. LEVEL OF EVIDENCE: Level 1, experimental study.

Impact of the Levonorgestrel-Releasing Intrauterine System on the Progression of Chlamydia trachomatis Infection to Pelvic Inflammatory Disease in a Baboon Model.

Background: Understanding the relationship between the levonorgestrel (LNG)-releasing intrauterine system (IUS) and sexually transmitted infections (STIs) is increasingly important as use of the LNG-IUS grows to include women at higher risk for STIs. This study assessed the impact of the LNG-IUS on development of Chlamydia trachomatis pelvic inflammatory disease, using a baboon model. Methods: Baboons with and those without the LNG-IUS were cervically inoculated with C. trachomatis and monitored daily, and cervical and fallopian tube swab specimens were collected weekly for C. trachomatis quantitation by nucleic acid amplification testing and culture. Vaginal swab specimens were collected for cytokine analysis, and serum samples were obtained for detection of C. trachomatis antibodies. Results: The LNG-IUS resulted in an increased C. trachomatis burden in the cervix, with the bacterial burden in the LNG-IUS group diverging from that in the non-LNG-IUS group by 6 weeks after infection. One of 7 baboons in the non-LNG-IUS group and 2 of 6 in the LNG-IUS group developed pelvic inflammatory disease, while 3 animals in each group met criteria suggestive of pelvic inflammatory disease. LNG-IUS increased baseline interleukin 8 levels but failed to further upregulate interleukin 8 during infection. In LNG-IUS recipients, early perturbations in the interleukin 1ß axis corresponded to decreased C. trachomatis clearance and increased T-helper type 2 immune responses. Conclusion: LNG-IUS use results in delayed clearance of C. trachomatis and might alter the reproductive tract immune environment.

Measurement of Mesoscale Conformational Dynamics of Freely Diffusing Molecules with Tracking FCS.

Few techniques are suited to probe the structure and dynamics of molecular complexes at the mesoscale level (∼100-1000 nm). We have developed a single-molecule technique that uses tracking fluorescence correlation spectroscopy (tFCS) to probe the conformation and dynamics of mesoscale molecular assemblies. tFCS measures the distance fluctuations between two fluorescently labeled sites within an untethered, freely diffusing biomolecule. To achieve subdiffraction spatial resolution, we developed a feedback scheme that allows us to maintain the molecule at an optimal position within the laser intensity gradient for fluorescence correlation spectroscopy. We characterized tFCS spatial sensitivity by measuring the Brownian end-to-end dynamics of DNA molecules as short as 1000 bp. We demonstrate that tFCS detects changes in the compaction of reconstituted nucleosome arrays and can assay transient protein-mediated interactions between distant sites in an individual DNA molecule. Our measurements highlight the applicability of tFCS to a wide variety of biochemical processes involving mesoscale conformational dynamics.

Impact of expedited partner therapy (EPT) implementation on chlamydia incidence in the USA.

OBJECTIVES: The diagnosis and treatment of Chlamydia trachomatis infection is important in preventing persistent or recurrent infection. Expedited partner therapy (EPT) is the favoured and supported method for STI treatment of the Centers for Disease Control and Prevention when the provider cannot be assured that all recent sexual partner(s) will seek therapy. EPT is legally permissible in 38 states and is endorsed by healthcare organisations to decrease the rates of chlamydia and gonorrhoea infection. Our study investigated the impact of EPT legal status (permissible, potentially allowable or prohibited) on C. trachomatis infection rates for each state. METHODS: Our ecological study modelled the number of reported chlamydia cases from 2000 to 2013 as a function of year, legal status and the interaction between year and legality. We used a negative binomial regression model that included state fixed effects (including the District of Columbia) to account for both the repeated measures per state and state-specific characteristics that could not be measured for inclusion in this study. The lagged number of C. trachomatis cases was included as a covariate and each state's total population for a given year was included in the model as an exposure parameter. States were designated Y (EPT permissible), N (EPT prohibited) and M (EPT potentially allowable), and the legal status of each state could vary over time. RESULTS: Each legal category saw an increase in the incidence rate of C. trachomatis infection, but on average, the incidence rate for states with prohibitive EPT legislation grew significantly faster over time compared with the rate for the states where EPT was permissible. The average increase in predicted incidence rates per year for states with Y, N and M legal status were 14.1 (95% CI (12.0 to 16.2)), 17.5 (95% CI (15.9 to 19.2)) and 16.8 (95% CI (15.0 to 18.6)) cases per 100 000 persons per year, respectively, when controlling for state-specific effects. CONCLUSIONS: Our model suggests that a lack of EPT legislation is associated with an increase in STI rates. States with potentially allowable EPT legislation as of 2013 (n=8) should consider permitting EPT as a component of a multipronged strategy for treatment of sexual partners to prevent C. trachomatis infection.

Direct imaging of RecA nucleation and growth on single molecules of SSB-coated ssDNA.

Escherichia coli RecA is the defining member of a ubiquitous class of DNA strand-exchange proteins that are essential for homologous recombination, a pathway that maintains genomic integrity by repairing broken DNA. To function, filaments of RecA must nucleate and grow on single-stranded DNA (ssDNA) in direct competition with ssDNA-binding protein (SSB), which rapidly binds and continuously sequesters ssDNA, kinetically blocking RecA assembly. This dynamic self-assembly on a DNA lattice, in competition with another protein, is unique for the RecA family compared to other filament-forming proteins such as actin and tubulin. The complexity of this process has hindered our understanding of RecA filament assembly because ensemble measurements cannot reliably distinguish between the nucleation and growth phases, despite extensive and diverse attempts. Previous single-molecule assays have measured the nucleation and growth of RecA--and its eukaryotic homologue RAD51--on naked double-stranded DNA and ssDNA; however, the template for RecA self-assembly in vivo is SSB-coated ssDNA. Using single-molecule microscopy, here we directly visualize RecA filament assembly on single molecules of SSB-coated ssDNA, simultaneously measuring nucleation and growth. We establish that a dimer of RecA is required for nucleation, followed by growth of the filament through monomer addition, consistent with the finding that nucleation, but not growth, is modulated by nucleotide and magnesium ion cofactors. Filament growth is bidirectional, albeit faster in the 5'→3' direction. Both nucleation and growth are repressed at physiological conditions, highlighting the essential role of recombination mediators in potentiating assembly in vivo. We define a two-step kinetic mechanism in which RecA nucleates on transiently exposed ssDNA during SSB sliding and/or partial dissociation (DNA unwrapping) and then the RecA filament grows. We further demonstrate that the recombination mediator protein pair, RecOR (RecO and RecR), accelerates both RecA nucleation and filament growth, and that the introduction of RecF further stimulates RecA nucleation.

Prevalence and geographical distribution of Papio hamadryas papillomavirus 1 (PhPV1) in Kenyan baboons.

Papio hamadryas papillomavirus (PhPV) 1, 2, and 3, are Alphapapillomaviruses that have been detected in Kenyan Olive baboons but the distribution is unknown. Therefore, cervical screening for PhPV1 was performed in baboons from various areas in Kenya using a nested polymerase chain reaction. The prevalence rate was 33%.

Laparoscopic Removal of a Retroperitoneal Hysteroscopic Microinsert Using Fluoroscopy.

Perforation during placement of hysteroscopic microinserts for permanent sterilization occurs in approximately .9% to 2.6% of women undergoing the procedure. Most of the time perforation results in intraperitoneal placement of the hysteroscopic microinsert requiring laparoscopy or laparotomy for removal of the device. Herein we present a case of hysteroscopic microinsert perforation with subsequent retroperitoneal identification of the device. This is the first such case to our knowledge of retroperitoneal identification and retrieval of a perforated device that required real-time fluoroscopy during laparoscopy.