ATPase cycle and DNA unwinding kinetics of RecG helicase

The superfamily 2 bacterial helicase, RecG, is a monomeric enzyme with a role in DNA repair by reversing stalled replication forks. The helicase must act specifically and rapidly to prevent replication fork collapse. We have shown that RecG binds tightly and rapidly to four-strand oligonucleotide junctions, which mimic a stalled replication fork. The helicase unwinds such DNA junctions with a step-size of approximately four bases per ATP hydrolyzed. To gain an insight into this mechanism, we used fluorescent stopped-flow and quenched-flow to measure individual steps within the ATPase cycle of RecG, when bound to a DNA junction. The fluorescent ATP analogue, mantATP, was used throughout to determine the rate limiting steps, effects due to DNA and the main states in the cycle. Measurements, when possible, were also performed with unlabeled ATP to confirm the mechanism. The data show that the chemical step of hydrolysis is the rate limiting step in the cycle and that this step is greatly accelerated by bound DNA. The ADP release rate is similar to the cleavage rate, so that bound ATP and ADP would be the main states during the ATP cycle. Evidence is provided that the main structural rearrangements, which bring about DNA unwinding, are linked to these states

DOES SPACE MATTER? AN INVESTIGATION OF THE SPATIAL VARIATION IN GESTATIONAL WEIGHT GAIN ASSOCIATIONS WITH COUNTRY OF BIRTH AMONG HISPANIC WOMEN AND ACCESS TO CARE IN TEXAS

Inadequate and excess gestational weight gain (GWG) are serious, but potentially preventable adverse pregnancy outcomes which affect as many as two-thirds of pregnant women in the United States. While location and space are associated with a variety of pregnancy outcomes, limited research has investigated potential spatial variation in GWG. The goal of this research is to improve the knowledge of how spatial geography is associated with GWG through secondary analyses of 70,000 to 160,000 birth certificate registry records for Texas mothers with a live birth delivery in 2014. Specifically, this research attempts to assess whether access to obstetrics and gynecologist (OBGYN) providers and country of birth among Hispanic women were associated with gestational weight gain. Overall, this research indicates that space is important in understanding GWG, though the significance of space depends on the studied risk factor. Analyses of women in the Houston-The Woodlands-Sugarland Metropolitan Statistical Area revealed that potential geographic access to OBGYN providers was not associated with inadequate or excess GWG; although, there was variation in the odds of inadequate or excess GWG in the area. Additionally, analyses of Hispanic women demonstrated statistically significant variation in the association between maternal county of birth and total GWG. Patterns indicated that foreign-born maternal birth compared to maternal birth in the United States is associated with increased total GWG along the Texas-Mexico border and in rural areas in Texas with a shift to less GWG along the Texas I-35 corridor and in northwest Texas. Consistent across the research was the importance of the association between prepregnancy weight and GWG. While space is important to understanding GWG, this research reveals that prepregnancy weight may be the key factor in controlling GWG. Few studies explore spatial variation in GWG and this was the first to explore variation within Texas which could show variation in studied maternal characteristics across the entire state. Public health researchers may utilize methods from this research as a template for incorporating spatial components into their research as space may improve the modelling process and elucidate the role of studied health characteristics, investigate the possibility of a threshold effect for geographic access to care, and explore the role of spatial variation in the Hispanic Paradox

The ATPase cycle of PcrA helicase and its coupling to translocation on DNA.

The superfamily 1 bacterial helicase PcrA has a role in the replication of certain plasmids, acting with the initiator protein (RepD) that binds to and nicks the double-stranded origin of replication. PcrA also translocates single-stranded DNA with discrete steps of one base per ATP hydrolyzed. Individual rate constants have been determined for the DNA helicase PcrA ATPase cycle when bound to either single-stranded DNA or a double-stranded DNA junction that also has RepD bound. The fluorescent ATP analogue 2'(3')-O-(N-methylanthraniloyl)ATP was used throughout all experiments to provide a complete ATPase cycle for a single nucleotide species. Fluorescence intensity and anisotropy stopped-flow measurements were used to determine rate constants for binding and release. Quenched-flow measurements provided the kinetics of the hydrolytic cleavage step. The fluorescent phosphate sensor MDCC-PBP was used to measure phosphate release kinetics. The chemical cleavage step is the rate-limiting step in the cycle and is essentially irreversible and would result in the bound ATP complex being a major component at steady state. This cleavage step is greatly accelerated by bound DNA, producing the high activation of this protein compared to the protein alone. The data suggest the possibility that ADP is released in two steps, which would result in bound ADP also being a major intermediate, with bound ADP.P(i) being a very small component. It therefore seems likely that the major transition in structure occurs during the cleavage step, rather than P(i) release. ATP rebinding could then cause reversal of this structural transition. The kinetic mechanism of the PcrA ATPase cycle is very little changed by potential binding to RepD, supporting the idea that RepD increases the processivity of PcrA by increasing affinity to DNA rather than affecting the enzymatic properties per se

American Myths, Legends, and Tall Tales: An Encyclopedia of American Folklore

Folklore has been a part of American culture for as long as humans have inhabited North America, and increasingly formed an intrinsic part of American culture as diverse peoples from Europe, Africa, Asia, and Oceania arrived. In modern times, folklore and tall tales experienced a rejuvenation with the emergence of urban legends and the growing popularity of science fiction and conspiracy theories, with mass media such as comic books, television, and films contributing to the retelling of old myths. This multi-volume encyclopedia will teach readers the central myths and legends that have formed American culture since its earliest years of settlement. Its entries provide a fascinating glimpse into the collective American imagination over the past 400 years through the stories that have shaped it. [From the Publisher]https://cupola.gettysburg.edu/books/1095/thumbnail.jp

ATPase mechanism of the 5'-3' DNA helicase, RecD2: evidence for a pre-hydrolysis conformation change

The superfamily 1 helicase, RecD2, is a monomeric, bacterial enzyme with a role in DNA repair, but with 5'-3' activity unlike most enzymes from this superfamily. Rate constants were determined for steps within the ATPase cycle of RecD2 in the presence of ssDNA. The fluorescent ATP analog, mantATP (2'(3')-O-(N-methylanthraniloyl)ATP), was used throughout to provide a complete set of rate constants and determine the mechanism of the cycle for a single nucleotide species. Fluorescence stopped-flow measurements were used to determine rate constants for adenosine nucleotide binding and release, quenched-flow measurements were used for the hydrolytic cleavage step, and the fluorescent phosphate biosensor was used for phosphate release kinetics. Some rate constants could also be measured using the natural substrate, ATP, and these suggested a similar mechanism to that obtained with mantATP. The data show that a rearrangement linked to Mg(2+) coordination, which occurs before the hydrolysis step, is rate-limiting in the cycle and that this step is greatly accelerated by bound DNA. This is also shown here for the PcrA 3'-5' helicase and so may be a general mechanism governing superfamily 1 helicases. The mechanism accounts for the tight coupling between translocation and ATPase activity

Ocean acidification promotes otolith growth and calcite deposition in gilthead sea bream (Sparus aurata) larvae

This work received funding from the Marine Alliance for Science and Technology for Scotland (MASTS) Small Grant initiative (SG330 and SG407). MASTS is funded by the Scottish Funding Council (grant reference HR09011) and contributing institutions. PBW would like to thank the Royal Society for the award of an Industry Fellowship. Financial support to CRB and JG were through the BIOACID (Biological Impacts of Ocean Acidification; Phase II) research programme.The effects of ocean acidification on otolith crystallization and growth rates were investigated in gilthead sea bream (Sparus aurata) larvae. Larvae were exposed to three different pH levels: pH8.2, pH7.7 and pH7.3 for a period of 18 days post-fertilization. For the first time, we demonstrate that pH has a significant impact on the carbonate polymorph composition, showing calcite in a significant percentage of individuals at low pH. Around 21% of the larvae exposed to pH7.3 showed irregular calcitic otoliths rather than commonly found round aragonitic otoliths. Calcitic otoliths showed a moderate level of heritability suggesting an important role of genetic factors. We also observed significantly larger otoliths in larvae reared at pH7.7 and pH7.3 compared to pH8.2 in both sagittae and lapilli. Our results demonstrate that otolith growth rates in gilthead sea bream larvae increase at low pH while a significant proportion of larvae are prone to the formation of calcitic otoliths at pH7.3.Publisher PDFPeer reviewe

Predicting the potential for spread of emerald ash borer ( Agrilus planipennis ) in Great Britain: What can we learn from other affected areas?

Funder: United Kingdom Government Department for Environment, Food and Rural AffairsSocietal Impact Statement: Emerald ash borer (EAB) is thought to have arrived in North America and European Russia at least 10 years prior to detection. Despite heightened awareness that EAB could invade Great Britain (GB), detection in the early stages of establishment is difficult, and initial symptoms might be mistaken for Chalara ash dieback. Our results suggest that if partial resistance to EAB in Fraxinus excelsior does not significantly dampen EAB population dynamics, then EAB could establish and spread across large parts of southern England within a relatively short time period; however, further northern spread may be limited by the relatively cool climate. Summary: The accidental introduction of emerald ash borer (EAB) to North America and European Russia in the 1990s has resulted in an ongoing crisis with millions of ash trees damaged and killed at immense economic and social cost. Improving our understanding of how rapidly the pest might spread should it enter Great Britain (GB) plays an essential part in planning for a potential outbreak. Two metrics are used to investigate the potential dynamics of EAB in GB: the observed rate of spread in the North American and Russian regions; and the relationship between degree days and emergence that may determine environmental suitability and whether the life cycle is univoltine or semivoltine. The pest is still spreading in North America and European Russia with an overall average rate of spread between 2002 and 2018 of approximately 50 km a year. Early detection of the pest is difficult, but a similar delay in detection to that in North America would result in a costly and hard to control outbreak. Comparison of degree days between regions suggests that a semivoltine life cycle is most likely in most areas of GB but spread maybe limited by the relatively cool climate in parts of GB. There are several potentially important differences in the biophysical environment in GB compared with North America and European Russia. However, the speed with which it has invaded these areas highlights the need for early surveillance and mitigations to minimise human‐mediated spread of this highly destructive pest

Movements of Spawning and Non-Spawning Shovelnose Sturgeon in the Missouri River Above Fort Peck Reservoir

During the last 40 yrs there has been a lack of pallid sturgeon (Scaphirhynchus albus) recruitment in the upper Missouri River (UMR). However, shovelnose sturgeon (Scaphirhynchus platorynchus) continue to exhibit recruitment in the UMR. Understanding the recruitment dichotomy between species is receiving much attention throughout their range. The objectives of this study were to identify the effects of varying discharge on spawning locations and spawning movements for pallid and shovelnose sturgeon. Two female pallid sturgeon, 32 gravid female shovelnose sturgeon, and 32 non-reproductively active female shovelnose sturgeon were radio tagged at three locations and tracked from 1 May to 5 July 2009. Unfortunately, no data are available for spawning pallid sturgeon movements because fish were not reproductively active. Upstream movement by gravid shovelnose sturgeon varied from 20 percent of the fish tagged at Judith Landing to 56 percent of the fish tagged at Coal Banks Recreation Area (CBRA). Mean maximum upstream movement of gravid shovelnose sturgeon varied from 35.7 km at CBRA to 87.9 km at Fred Robinson Bridge (FRB), mean maximum downstream movement varied from 24.9 km at FRB to 80.3 km at CBRA. Reproductively inactive shovelnose sturgeon exhibited lower mean maximum movements than reproductively active fish (mean maximum distance 5.7 km). Shovelnose sturgeon in the UMR exhibit both upstream and downstream movements prior to spawning and are using several spawning areas. Thus, maintaining spawning habitat throughout a regulated river is important with regard to shovelnose sturgeon conservation

Transcatheter Closure of Paravalvular Defects Using a Purpose-Specific Occluder

ObjectivesThis study sought to describe a method of paravalvular leak closure using a purpose-specific occlusion device.BackgroundTranscatheter closure of paravalvular leaks has been hampered by technical challenges, the limitations of available imaging modalities, and the lack of closure devices specifically designed for this purpose.MethodsPatients with severe symptomatic paravalvular regurgitation at high risk for repeat surgery underwent transcatheter leak closure. Both left ventricular puncture and retrograde transfemoral approaches were used with fluoroscopic and 3-dimensional transesophageal guidance. A purpose-specific occluder (Vascular Plug III, AGA Medical Corp., Plymouth, Minnesota) was used.ResultsFive patients with severe prosthetic mitral and aortic paravalvular leaks underwent attempted closure. Implantation of the device was successfully accomplished in all. In 1 patient, the plug interfered with closure of a mechanical valve leaflet and was removed and replaced with an alternate device. Complications included pericardial bleeding in 2 patients with a transapical approach. There was no procedural mortality. At a median follow-up of 191 days (interquartile range [IQR] 169 to 203 days) all patients were alive. New York Heart Association functional class fell from 4 (IQR 3 to 4) to 2 (IQR 2 to 3), hemoglobin rose from 89 g/l (IQR 87 to 108 g/l) to 115 g/l (IQR 104 to 118 g/l), creatinine fell from 109 μmol/l (IQR 106 to 132 μmol/l) to 89 μmol/l (IQR 89 to 126 μmol/l). Median echocardiographic follow-up at 58 days (IQR 56 to 70 days) reported residual regurgitation to be reduced from grade 4 to grade 2 (IQR 1.5 to 2.25).ConclusionsClosure of mitral and aortic prosthetic paravalvular leaks with the Vascular Plug III using either a transapical (mitral) or a retrograde (aortic) approach appears promising