Flap Endonuclease Disengages Dna2 Helicase/Nuclease from Okazaki Fragment Flaps

Okazaki fragments contain an initiator RNA/DNA primer that must be removed before the fragments are joined. In eukaryotes, the primer region is raised into a flap by the strand displacement activity of DNA polymerase {delta}. The Dna2 helicase/nuclease and then flap endonuclease 1 (FEN1) are proposed to act sequentially in flap removal. Dna2 and FEN1 both employ a tracking mechanism to enter the flap 5' end and move toward the base for cleavage. In the current model, Dna2 must enter first, but FEN1 makes the final cut at the flap base, raising the issue of how FEN1 passes the Dna2. To address this, nuclease-inactive Dna2 was incubated with a DNA flap substrate and found to bind with high affinity. FEN1 was then added, and surprisingly, there was little inhibition of FEN1 cleavage activity. FEN1 was later shown, by gel shift analysis, to remove the wild type Dna2 from the flap. RNA can be cleaved by FEN1 but not by Dna2. Pre-bound wild type Dna2 was shown to bind an RNA flap but not inhibit subsequent FEN1 cleavage. These results indicate that there is a novel interaction between the two proteins in which FEN1 disengages the Dna2 tracking mechanism. This interaction is consistent with the idea that the two proteins have evolved a special ability to cooperate in Okazaki fragment processing

Dynamic removal of replication protein A by Dna2 facilitates primer cleavage during Okazaki fragment processing in Saccharomyces cerevisiae

Eukaryotic Okazaki fragments are initiated by an RNA/DNA primer, which is removed before the fragments are joined. Polymerase d displaces the primer into a flap for processing. Dna2 nuclease/helicase and flap endonuclease 1 (FEN1) are proposed to cleave the flap. The single-stranded DNA binding protein, replication protein A (RPA), governs cleavage activity. Flap-bound RPA inhibits FEN1. This necessitates cleavage by Dna2, which is stimulated by RPA. FEN1 then cuts the remaining RPA-free flap to create a nick for ligation. Cleavage by Dna2 requires that it enter the 5'-end and track down the flap. Since Dna2 cleaves the RPA-bound flap, we investigated the mechanism by which Dna2 accesses the protein-coated flap for cleavage. Using a nuclease-defective Dna2 mutant, we showed that just binding of Dna2 dissociates the flap-bound RPA. Facile dissociation is specific to substrates with a genuine flap, and will not occur with an RPA-coated single strand. We also compared the cleavage patterns of Dna2 with and without RPA to better define RPA stimulation of Dna2. Stimulation derived from removal of DNA folding in the flap. Apparently, coordinated with its dissociation, RPA relinquishes the flap to Dna2 for tracking in a way that does not allow flap structure to reform. We also found that RPA strand melting activity promotes excessive flap elongation, but it is suppressed by Dna2-promoted RPA dissociation. Overall, results indicate that Dna2 and RPA coordinate their functions for efficient flap cleavage and preparation for FEN1

Wildbook: Crowdsourcing, computer vision, and data science for conservation

Photographs, taken by field scientists, tourists, automated cameras, and incidental photographers, are the most abundant source of data on wildlife today. Wildbook is an autonomous computational system that starts from massive collections of images and, by detecting various species of animals and identifying individuals, combined with sophisticated data management, turns them into high resolution information database, enabling scientific inquiry, conservation, and citizen science. We have built Wildbooks for whales (flukebook.org), sharks (whaleshark.org), two species of zebras (Grevy's and plains), and several others. In January 2016, Wildbook enabled the first ever full species (the endangered Grevy's zebra) census using photographs taken by ordinary citizens in Kenya. The resulting numbers are now the official species census used by IUCN Red List: http://www.iucnredlist.org/details/7950/0. In 2016, Wildbook partnered up with WWF to build Wildbook for Sea Turtles, Internet of Turtles (IoT), as well as systems for seals and lynx. Most recently, we have demonstrated that we can now use publicly available social media images to count and track wild animals. In this paper we present and discuss both the impact and challenges that the use of crowdsourced images can have on wildlife conservation.Comment: Presented at the Data For Good Exchange 201

An epidemiologic profile of pediatric concussions: Identifying urban and rural differences

BACKGROUND: The objective of this study was to describe the epidemiology of concussions presenting to the emergency department (ED). METHODS: A retrospective cohort of concussions for pediatric (age G 18 years) patients treated in the ED of a regional pediatric Level 1 trauma center from 2006 to 2011 was examined. Descriptive and geographic analyses were completed, with comparisons by age groups and residence (urban/rural). RESULTS: There were a total of 2,112 treated pediatric concussions. Two thirds of the concussions occurred in males (67%), with a median age of 13 years (interquartile range [IQR], 6). Nearly half of the pediatric concussions were sports related (48%); 36% of these concussions were from hockey. Significant differences were found in the distribution of the mechanism of injury across age groups (p G 0.001). Falls were most prevalent among young children, and sports concussions, for children 10 years and older. Two fifths of concussions occurred during winter months. Discharge disposition significantly differed by age ( p G 0.001), with home discharge increasing with age up to 14 years. There were a total of 387 rural (19%) and 1,687 urban (81%) concussed patients, for a mean ED concussion visit rate of 2.2 per 1,000 and 3.5 per 1,000, respectively. Rural patients were older (14 [IQR, 6] vs. 13 [IQR, 6], p = 0.019] and sustained 2.5 times more concussions from a motor vehicle crash compared with urban youth patients ( p G 0.001). CONCLUSION: Males in early adolescence are at highest risk for concussion, particularly from sport-related activities. Urban and rural children have differences in their etiology and severity of concussions. Concussions are predictable, and their prevention should be targeted based on epidemiologic and environmental data

Methods for Estimating Kidney Disease Stage Transition Probabilities Using Electronic Medical Records

Chronic diseases are often described by stages of severity. Clinical decisions about what to do are influenced by the stage, whether a patient is progressing, and the rate of progression. For chronic kidney disease (CKD), relatively little is known about the transition rates between stages. To address this, we used electronic health records (EHR) data on a large primary care population, which should have the advantage of having both sufficient follow-up time and sample size to reliably estimate transition rates for CKD. However, EHR data have some features that threaten the validity of any analysis. In particular, the timing and frequency of labratory values and clinical measurements are not determined a priori by research investigators, but rather, depend on many factors, including the current health of the patient. We developed an approach for estimatating CKD stage transition rates using hidden Markov models (HMMs), when the level of information and observation time vary among individuals. To estimate the HMMs in a computationally manageable way, we used a “discretization” method to transform daily data into intervals of 30 days, 90 days, or 180 days. We assessed the accuracy and computation time of this method via simulation studies. We also used simulations to study the effect of informative observation times on the estimated transition rates. Our simulation results showed good performance of the method, even when missing data are non-ignorable. We applied the methods to EHR data from over 60,000 primary care patients who have chronic kidney disease (stage 2 and above). We estimated transition rates between six underlying disease states. The results were similar for men and women

Motor, not visual, encoding of potential reach targets

SummaryWe often encounter situations in which there are multiple potential targets for action, as when, for example, we hear the request “could you pass the …” at the dinner table. It has recently been shown that, in such situations, activity in sensorimotor brain areas represents competing reach targets in parallel prior to deciding between, and then reaching towards, one of these targets [1]. One intriguing possibility, consistent with the influential notion of action ‘affordances’ [2], is that this activity reflects movement plans towards each potential target [3]. However, an equally plausible explanation is that this activity reflects an encoding of the visual properties of the potential targets (for example, their locations or directions), prior to any target being selected and the associated movement plan being formed. Notably, previous work showing spatial averaging behaviour during reaching, in which initial movements are biased towards the midpoint of the spatial distribution of potential targets [4–6], remains equally equivocal concerning the motor versus visual encoding of reach targets. Here, using a rapid reaching task that disentangles these two competing accounts, we show that reach averaging behaviour reflects the parallel encoding of multiple competing motor plans. This provides direct evidence for theories proposing that the brain prepares multiple available movements before selecting between them [3]

An evidence-based method for targeting an abusive head trauma prevention media campaign and its evaluation

BACKGROUND: A triple-dose abusive head trauma (AHT) prevention program (Period of PURPLE Crying) was implemented. The third dose consisted of an education media campaign. The study objectives were to describe the qualitative and spatial methods developed to target AHT prevention and to evaluate this campaign. METHODS: A questionnaire on the level of importance of factors, rated on a 7-point Likert scale, was distributed to a panel of experts to determine the best advertising locations. Ranked factors were used to create weights for statistical modeling and mapping within a Geographic Information Systems to determine optimal ad locations. The media campaign was evaluated via a telephone survey of randomly selected households. RESULTS: The survey found locations of new families, high population density, and high percentage of lone parents to be the most important factors for selecting billboard sites. Spatial analysis revealed six areas that ranked highest in our factors. Five billboards, four media posters, and six transit shelters were selected for our advertisements. A population-based telephone survey revealed that 23% of respondents knew the campaign. Nearly half (42%) heard the radio public service announcements, and 9% saw billboards. CONCLUSION: Extending primary prevention efforts to the public helps to create a cultural change in the way inconsolable crying, the trigger for AHT, is viewed. With the use of ranked factors and Geographic Information Systems, geographic locations with high visibility and specific risk factors for AHTwere identified for targeting the campaign, facilitating the likelihood that our message was reaching the population in greatest need

Significance of the dissociation of Dna2 by flap endonuclease 1 to Okazaki fragment processing in Saccharomyces cerevisiae

Okazaki fragments are initiated by short RNA/DNA primers, which are displaced into flap intermediates for processing. Flap endonuclease 1 (FEN1) and Dna2 are responsible for flap cleavage. Replication protein A (RPA)-bound flaps inhibit cleavage by FEN1 but stimulate Dna2, requiring that Dna2 cleaves prior to FEN1. Upon cleavage, Dna2 leaves a short flap, which is then cut by FEN1 forming a nick for ligation. Both enzymes require a flap with a free 5'-end for tracking to the cleavage sites. Previously, we demonstrated that FEN1 disengages the tracking mechanism of Dna2 to remove it from the flap. To determine why the disengagement mechanism evolved, we measured FEN1 dissociation of Dna2 on short RNA and DNA flaps, which occur during flap processing. Dna2 tracked onto these flaps but could not cleave, presenting a block to FEN1 entry. However, FEN1 disengaged these nonproductively bound Dna2 molecules, proceeding on to conduct proper cleavage. These results clarify the importance of disengagement. Additional results showed that flap substrate recognition and tracking by FEN1, as occur during fragment processing, are required for effective displacement of the flap-bound Dna2. Dna2 was recently shown to dissociate flap-bound RPA, independent of cleavage. Using a nuclease-defective Dna2 mutant, we reconstituted the sequential dissociation reactions in the proposed RPA/Dna2/FEN1 pathway showing that, even without cutting, Dna2 enables FEN1 to cleave RPA-coated flaps. In summary, RPA, Dna2, and FEN1 have evolved highly coordinated binding properties enabling one protein to succeed the next for proper and efficient Okazaki flap processing

Dna2 is a structure-specific nuclease, with affinity for 5'-flap intermediates

Dna2 is a nuclease/helicase with proposed roles in DNA replication, double-strand break repair and telomere maintenance. For each role Dna2 is proposed to process DNA substrates with a 5'-flap. To date, however, Dna2 has not revealed a preference for binding or cleavage of flaps over single-stranded DNA. Using DNA binding competition assays we found that Dna2 has substrate structure specificity. The nuclease displayed a strong preference for binding substrates with a 5'-flap or some variations of flap structure. Further analysis revealed that Dna2 recognized and bound both the single-stranded flap and portions of the duplex region immediately downstream of the flap. A model is proposed in which Dna2 first binds to a flap base, and then the flap threads through the protein with periodic cleavage, to a terminal flap length of ~5 nt. This resembles the mechanism of flap endonuclease 1, consistent with cooperation of these two proteins in flap processing