Analysis Of The Role Of Dnaa Recognition Site Position In Directing Assembly Of The Pre-Replicative Complex In Escherichia Coli

Prior to initiating new rounds of DNA replication, all cells assemble pre-replicative complexes (pre-RC) consisting of initiator proteins and regions of DNA termed origins of replication. In the bacterial model system, E. coli, multiple copies of the initiator protein, DnaA,assemble into a complex with the unique chromosomal replication origin, oriC, to produce a pre-RC that unwinds the DNA helix, preparing the origin for the new replication forks. Nine base pair (bp) low affinity DnaA recognition sites are distinctly arrayed in each half of oriC and both arrays are bounded by high affinity recognition sites. Although it is known that DnaA does not interact at low affinity site arrays without assistance from DnaA occupying a proximally positioned high affinity site, it remains unclear why this particular arrangement of high and low affinity sites exists. Furthermore, despite the fact that DnaA is a highly conserved protein among all bacteria, the numbers and arrangements of high and low affinity sites in oriC varies considerably among different bacterial types. In order to understand the reason for this diversity, more information about E. coli oriC geography and its relationship to pre-RC assembly and origin function is required. In the studies reported here, two specific questions about E. coli oriC geography were addressed: 1) are all three high affinity sites required for oriC function, and 2) is the nucleotide spacing between recognition sites an inflexible feature. To answer these questions, mutagenized versions of E. coli oriC on chromosomes were evaluated for function in vivo, and DNA fragments containing different spacing and arrangements of high and low affinity recognition sites were examined by electrophoretic mobility shift assays (EMSA), high resolution DNA footprints, and DNA unwinding assays.When inserted into E. coli as replacements for the wild-type chromosomal origin, mutant versions of oriC lacking any two high affinity recognition sites (R1, R2, or R4) were inactive, but origins carrying only one inactivated high affinity DnaA recognition site retained function no matter which site was altered. This finding contradicts previous reports that the R1siteis essential for oriC function, although loss of either R1 or R4 binding caused reduced origin activity and loss of initiation synchrony.Although it appears that the requirement for high affinity DnaA binding is not stringent for origin activity, a novel phenotype for high affinity site-deficient mutants was identified. Supercoiled oriC DNA is not normally unwound at low levels of DnaA, but spontaneous DNA unwinding of every high affinity binding mutant was detected under these conditions. A model is proposed whereby oriC DNA is normally constrained by DnaA during the cell cycle to prevent spontaneous initiations and constraint requires the cooperative interactions among the distantly spaced DnaA bound to high affinity sites as an origin recognition complex. These interactions, presumably through the proper positioning of domain I-domain I contacts,would generate multiple DNA loops, analogous to the wrapping of DNA around nucleosomes in eukaryotes.Further contraction and stabilization of these loops by additional DnaA occupying arrayed low affinity sites would then produce the torsional stress required to separate DNA strands within the A-T rich DNA Unwinding Element (DUE)at the left side of oriC.Electrophoretic mobility shift assays (EMSA)with DNA fragments containing differently spaced DnaA recognition sites revealed that very small changes in base pair spacing between low-low or high-low affinity recognition sites resulted in dramatic changes in cooperative DnaA binding required for site occupation.Optimal binding at any two sites was detected at 2 bp spacing, but was diminished as single bases were added.Surprisingly, reducing the 2 bp spacing also diminished DnaA binding suggesting that some structural interference exists between adjacent DnaA molecules when they are brought too close together. These observations are consistent with specific positioning of DnaA recognition sites in E. coli oriC for correct origin function and even slight alterations in these positions have an impact. While it remains difficult to predict where DnaA recognition sites will be placed in the replication origin of any particular bacteria type, the knowledge gained from the studies presented here establishes a rule set that include the requirement for the arrangement of high affinity DnaA recognition sites to, at an early stage in the cell cycle, topologically constrain the oriC in an origin recognition complex and the requirement for additional recognition sites that convert the constrained complex into one that imparts sufficient torsional stress to unwind oriC

Taqman Real-Time PCR Detects Avipoxvirus DNA in Blood of Hawaìi `Amakihi (Hemignathus virens)

Margaret E. M. Farias et al...Background Avipoxvirus sp. is a significant threat to endemic bird populations on several groups of islands worldwide, including Hawaìi, the Galapagos Islands, and the Canary Islands. Accurate identification and genotyping of Avipoxvirus is critical to the study of this disease and how it interacts with other pathogens, but currently available methods rely on invasive sampling of pox-like lesions and may be especially harmful in smaller birds. Methodology/Principal Findings Here, we present a nested TaqMan Real-Time PCR for the detection of the Avipoxvirus 4b core protein gene in archived blood samples from Hawaiian birds. The method was successful in amplifying Avipoxvirus DNA from packed blood cells of one of seven Hawaiian honeycreepers with confirmed Avipoxvirus infections and 13 of 28 Hawaìi `amakihi (Hemignathus virens) with suspected Avipoxvirus infections based on the presence of pox-like lesions. Mixed genotype infections have not previously been documented in Hawaìi but were observed in two individuals in this study. Conclusions/Significance We anticipate that this method will be applicable to other closely related strains of Avipoxvirus and will become an important and useful tool in global studies of the epidemiology of Avipoxvirus.Funding for this study was provided by: U.S. Geological Survey, Pacific Island Ecosystems Research Center (biology.usgs.gov/pierc/); U.S. Geological Survey Wildlife (biology.usgs.gov/wter/) and Invasive Species (biology.usgs.gov/invasive/) Programs; National Science Foundation (DEB0083944, www.nsf.gov); NIH/NCRR IDeA Networks of Biomedical Research Excellence (INBRE), P20RR016467 (http://www.ncrr.nih.gov/research_infrast​ructure/institutional_development_award/​idea_networks_of_biomedical_research_exc​ellence/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewe

Major histocompatibility complex class II gene diversity and tolerance to avian malaria in Hawaiian honeycreepers (Drepanidinae) (99.15)

Abstract Susceptibility to avian malaria differs both within and between species of Hawaiian honeycreepers. Due to their critical function in adaptive immunity, proteins encoded by Mhc genes may be key in the development of tolerance to Plasmodium relictum in low elevation populations of `amakihi (Hemignathus virens). To evaluate gene copy number, 263 Mhc class II peptide-binding region clones were sequenced from a captive family of `amakihi. Phylogenetic analysis suggests a minimum of 10 Mhc class II genes with detection of 35 unique alleles. Species comparisons (total 503 clones, eight birds/species) reveal that Mhc diversity is highest in `amakihi [d = 0.085(0.015)] with 98 distinct alleles detected (average 13.5/bird), followed by `apapane (Himatione sanguinea) [d = 0.068(0.012)] with 66 distinct alleles detected (average 11/bird) and in a highly susceptible species, the i`iwi (Vestiaria coccinea) diversity appears lowest with d = 0.057(0.011) and 66 distinct alleles detected (average 10.6/bird). Within `amakihi, Mhc sequences from birds from low elevation (56 distinct alleles, average 14.25 alleles/bird) and high elevation (50 distinct alleles, average 12.75 alleles/bird) subpopulations appear distinct with detection of only one shared allele. Microarray-based population level analysis (~1,000 individuals) of Mhc diversity in `amakihi detected at least one allele with a significantly higher frequency in low elevation populations than mid or high elevation subpopulations.</jats:p

Contamination of the Mini C-Arm During Foot and Ankle Surgery

Background: Many orthopedic surgeries utilize intraoperative fluoroscopy. The mini C-arm is an advantageous device as it can be easily used without the need for a dedicated radiology technician. However, there are concerns that the mini C-arm may represent a potential source of contamination and subsequent postoperative infection. Previous investigations of standard C-arm drapes have shown high rates of contamination. Similar contamination rates would be even more concerning for the mini C-arm as it requires physically maneuvering the machine. This study aimed to determine the rate of mini C-arm drape contamination and identify high-risk areas. Methods: Fifty foot and ankle surgeries requiring the use of mini C-arm fluoroscopy were included. Eight locations on the mini C-arm drape were sampled at the conclusion of each procedure. Culture Q-swabs were used for sampling defined locations. Swab samples were then assessed for bacterial growth on a 5% blood agar plate using a semiquantitative technique. Results: In 70% of surgical cases, contamination was observed in at least 1 location. Six of the 8 evaluated locations were found to have significantly higher contamination in comparison with their corresponding negative controls (Mann-Whitney U test, P &lt; .05). The “outer portion of the upper arm” (location 1) exhibited bacteria growth in 26% ( P &lt; .0001) of cases. The “superior portion of the x-ray source” (location 2) exhibited growth in 30% ( P &lt; .0001) of cases. These were the highest-risk areas for contamination and were both significantly more likely to be involved than the “inferior portion of the x-ray source” and “superior portion of the beam receiver,” locations 4 and 5, respectively. Fourteen percent (7/50) C-arm cases and 1.72% (1/58) Achilles tendon surgery control cases developed surgical site infection ( P = .0234; OR, 9.27). Conclusion: Bacterial contamination of the mini C-arm drape was found to be common after foot and ankle procedures. Contamination was more prevalent on the outer ring areas of the C-arm, both at the emitter and receiver. Level of Evidence: Level III. </jats:sec

Evidence for the Will Rogers Phenomenon in Migration of Employees to Managed Care Plans

Employees have increasing opportunities to enroll in managed care plans, and employers tend to favor these plans because of their lower costs. However, lower costs may be the result of selection of healthier patients into managed care plans. This study measured differences in health care utilization across an indemnity plan and a managed care plan, and for all employees together. We found that apparent increases in utilization in both indemnity and managed care plans disappeared when the plans were viewed together, reflecting the migration of sicker patients from indemnity plans to managed care plans

Anatomic Structures at Risk During Posterior to Anterior Percutaneous Screw Fixation of Posterior Malleolar Fractures: A Cadaveric Study

Background. There are no established guidelines for fixation of posterior malleolus fractures (PMFs). However, fixation of PMFs appears to be increasing with growing evidence demonstrating benefits for stability, alignment, and early functional outcomes. The purpose of this study was to determine the risk to anatomic structures utilizing a percutaneous technique for posterior to anterior (PA) screw fixation of PMFs. Methods. Percutaneous PA screw placement was carried out on 10 fresh frozen cadaveric ankles followed by dissection to identify soft tissue and neurovascular structures at risk. The distance from the guidewire to each anatomic structure of interest was measured. The correlation between the mean distances from the guidewire to each structure was calculated. Results. The sural nerve was directly transected in 1/10 specimens (10%) and in contact with the wire in a second specimen (10%). There was a significant correlation between the proximity of the guidewire to the apex of Volkmann’s tubercle and its proximity to the sural nerve. The flexor hallucis longus (FHL) muscle belly was perforated by the guidewire 40% of the time but was not tethered or entrapped by the screw. Conclusions. Percutaneous PA screw placement is a safe technique which can be improved with several modifications. A mini-open technique is recommended to protect the sural nerve. There may be potential for tethering of the FHL with use of a washer or large screw head. Risk to the anterior and posterior neurovascular bundles is minimal. Levels of Evidence: Level V </jats:p

Contamination Profile of the Mini C-Arm Fluoroscopy Unit in Orthopedic Surgery

Category: Other; Ankle Introduction/Purpose: Fluoroscopy is critical for orthopaedic surgery. The mini C-arm is popular among foot and ankle surgeons owing ease of easy, maneuverability, and cost effectiveness. However, there are concerns that the mini C-arm may represent a potential source of contamination and increasing the risk of postoperative infection. Evaluation of large c-arm drapes have shown high rates of contamination. However, the mini C-arm may be even more concerning due to how it is handled. The mini c-arm is continuously manipulated by the surgical team who then return to the surgical field carrying with them any potential cross-contamination.Objective: We hypothesize that there is significant contamination of the drape, and that specific locations of the drape are at higher risk. Methods: 50 foot and ankle surgery cases which required the use of mini C-arm fluoroscopy were included. Eight locations on the mini C-arm drape were chosen for sampling. Locations were defined based on commonly touched areas on the drape by the surgical team, and their proximity to the non-sterile field. Negative controls were obtained by separately sampling an additional 20 mini C-arm drapes after draping c-arm and waiting for average surgery time. Positive controls were obtained by sampling non- sterile surfaces within the OR.Culture Q-swabs were used for sampling defined locations, using an established semiquantitative technique. Plates were incubated at 37 °C for 48 hours. Based on the number of quadrants on plate where colony growth was observed, bacterial growth was graded on a scale of 0, 1+, 2+, 3+, or 4+. Contamination threshold was defined as any growth pattern of 1+ or higher. Results: Figure 1 shows the contamination rates of the sampled areas of the c-arm drape. Contamination was noted in 70% of the cases for at least one area, and in two or more areas in 38% of cases.Contamination rates were significantly higher on defined locations in comparison with their relative negative control, except for locations 4 and 5. (P&lt;0.05)The most frequently contaminated location was location #2 (30%) followed by Location 1 (26%), and then location 8 (22%). (P&lt;0.05) Conclusion: Bacterial contamination of the drape was found to be common (70% of cases) after foot and ankle surgery. Contamination was more common on the superior and inferior aspects of the mini c-arm. This correlates to the area that is most commonly manipulated by the surgical team to position the arms of the c-arm. This information can be utilized by surgeons to identify and avoid high risk areas and, in high risk cases, they may consider changing gloves or re-draping the mini c-arm part way through the case. </jats:sec

Anatomic Structures at Risk during Posterior to Anterior Percutaneous Screw Fixation of Posterior Malleolar Fractures: A Cadaveric Study

Category: Ankle; Trauma Introduction/Purpose: Posterior malleolus fractures (PMFs) are typically associated with trimalleolar ankle fractures and have been reported to occur in 10-40% of ankle fractures. The PM may be fixed by direct ORIF or indirect percutaneous reduction. Direct reduction via a posterolateral approach is required for larger fragments, which requires prone or lateral decubitus positioning. Indirect reduction and fixation with anterior to posterior (AP) screws remains the most common method, in which screw threads are not entirely across the fracture site to allow interfragmentary compression.Objective: Determine the risk to anatomic structures utilizing a percutaneous technique for posterior to anterior (PA) screw fixation. It is advantageous as it places all the threads across the fracture site even for small fragments and can be performed in the supine position. Methods: 10 fresh-frozen, morphologically normal cadaver lower leg. Under fluoroscopic guidance, 1.35 mm Kirschner wire was inserted percutaneously from anteromedial to posterolateral. Guidewire was inserted from a starting point just medial to the tibialis anterior tendon and aimed at a point just lateral to the Achilles tendon. Small skin incision was made over the wire posteriorly, displacing the Achilles tendon medially as required. The wire was then over-drilled in posterior to anterior direction and replaced with a 4.0 mm partially threaded cannulated screw directed posterior to anterior. Each specimen was dissected, and adjacent soft tissue and neurovascular structures were identified. The distance from the guidewire to each anatomic structure of interest was measured. Descriptive analysis and the correlation between the mean distances from the guidewire to each structure was calculated using SPSS version 26. Results: The sural nerve was directly transected in 1/10 specimens (10%) and in contact with the wire in a second specimen (10%). There was a significant correlation between the proximity of the guidewire to the apex of Volkmann’s tubercle and its proximity to the sural nerve.(r= 0.705, p = 0.034) The flexor hallucis longus (FHL) muscle belly was perforated by the guidewire 40% of the time but was not tethered or entrapped by the screw. The neurovascular bundles were safely away from the wire in all cases. The lateral 1-2 mm of the Achilles tendon was pierced by the guidewire 20% of the time. Although suboptimal, these injuries may be of little clinical consequence, and the screw passed without tethering the tendon in all cadaveric specimens. Conclusion: Percutaneous PA screw placement is a safe technique which can be improved with several modifications. A mini- open technique is recommended to protect the sural nerve. The surgeon may consider aiming slightly more medial and closer to the Achilles tendon in order to avoid injury to the sural nerve. It is also advisable to use a soft tissue guide when over-drilling the guidewire. There may be potential for tethering of the FHL with use of a washer or large screw head. Risk to the anterior and posterior neurovascular bundles is minimal. </jats:sec