Direct observation of topoisomerase IA gate dynamics

Type IA topoisomerases cleave single-stranded DNA and relieve negative supercoils in discrete steps corresponding to the passage of the intact DNA strand through the cleaved strand. Although type IA topoisomerases are assumed to accomplish this strand passage via a protein-mediated DNA gate, opening of this gate has never been observed. We developed a single-molecule assay to directly measure gate opening of the Escherichia coli type IA topoisomerases I and III. We found that after cleavage of single-stranded DNA, the protein gate opens by as much as 6.6 nm and can close against forces in excess of 16 pN. Key differences in the cleavage, ligation, and gate dynamics of these two enzymes provide insights into their different cellular functions. The single-molecule results are broadly consistent with conformational changes obtained from molecular dynamics simulations. These results allowed us to develop a mechanistic model of interactions between type IA topoisomerases and single-stranded DNA

Ten practical realities for institutional animal care and use committees when evaluating protocols dealing with fish in the field

Institutional Animal Care and Use Committee’s (IACUCs) serve an important role in ensuring that ethical practices are used by researchers working with vertebrate taxa including fish. With a growing number of researchers working on fish in the field and expanding mandates of IACUCs to regulate field work, there is potential for interactions between aquatic biologists and IACUCs to result in unexpected challenges and misunderstandings. Here we raise a number of issues often encountered by researchers and suggest that they should be taken into consideration by IACUCs when dealing with projects that entail the examination of fish in their natural environment or other field settings. We present these perspectives as ten practical realities along with their implications for establishing IACUC protocols. The ten realities are: (1) fish are diverse; (2) scientific collection permit regulations may conflict with IACUC policies; (3) stakeholder credibility and engagement may constrain what is possible; (4) more (sample size) is sometimes better; (5) anesthesia is not always needed or possible; (6) drugs such as analgesics and antibiotics should be prescribed with care; (7) field work is inherently dynamic; (8) wild fish are wild; (9) individuals are different, and (10) fish capture, handling, and retention are often constrained by logistics. These realities do not imply ignorance on the part of IACUCs, but simply different training and experiences that make it difficult for one to understand what happens outside of the lab where fish are captured and not ordered/purchased/reared, where there are engaged stakeholders, and where there is immense diversity (in size, morphology, behaviour, life-history, physiological tolerances) such that development of rigid protocols or extrapolation from one species (or life-stage, sex, size class, etc.) to another is difficult. We recognize that underlying these issues is a need for greater collaboration between IACUC members (including veterinary professionals) and field researchers which would provide more reasoned, rational and useful guidance to improve or maintain the welfare status of fishes used in field research while enabling researchers to pursue fundamental and applied questions related to the biology of fish in the field. As such, we hope that these considerations will be widely shared with the IACUCs of concerned researchers

Effects of acclimation temperature on critical thermal limits and swimming performance of the state-endangered bigeye chub Hybopsis amblops

Thermal stress can directly affect the survival of fishes and indirectly impact fish populations through several processes, including impaired swimming performance. Bigeye chub Hybopsis amblops is a state-endangered species in Illinois and is disappearing in the northern portion of its native range in North America. Limited temperature tolerance information exists on this species. The aim of this study was to define the impacts of 2 acclimation temperatures on the performance and behavior of bigeye chub. To accomplish this, we conducted 2 assays: critical thermal maximum (CTmax) testing for upper thermal tolerance limits, and swimming performance testing for critical swimming speed (Ucrit) and burst swimming ability. With a 5°C acclimation temperature increase from 21 to 26°C, the CTmax of bigeye chub increased from 32.8 ± 0.4°C to 36.4 ± 0.9°C. Ucrit was not different across acclimation temperatures, and fish from both acclimation groups could swim up to over 10 body lengths (BL) s-1. Burst swimming duration also did not differ statistically across groups, but bigeye chub from the 26°C group swam 27% longer in duration relative to fish from the 21°C group. Results from this study can help guide the protection and restoration of bigeye chub populations from thermal stressors

Is there a pace-of-life syndrome linking boldness and metabolic capacity for locomotion in bluegill sunfish?

The concept of behavioural syndromes (i.e. correlations between behavioural traits) has provided an important framework for understanding individual variation in animal behaviour and its link to individual variation in physiology and life-history traits. The pace-of-life syndrome concept posits that behavioural, physiological and life-history traits coevolve in response to correlated selection pressures, and therefore predicts a positive correlation between boldness (i.e. exploration and risk taking) and metabolic capacity for locomotor performance in individuals. We tested for a pace-of-life syndrome linking boldness and metabolic capacity for locomotor activity in juvenile bluegill sunfish, Lepomis macrochirus. Individual fish were screened and classified as bold or shy using an established refuge emergence test. Subsequently, the aerobic and anaerobic metabolisms of bold and shy individuals were quantified using respirometry and by measuring the metabolic by-products of white muscle anaerobic glycolysis following exhaustive exercise, respectively. Bold fish demonstrated 25% greater metabolic scope for activity (i.e. aerobic capacity) than shy fish, which was attributable to a 15% greater maximum metabolic rate. However, there was no significant difference in resting metabolic rate or anaerobic energy expenditure (i.e. anaerobic capacity) between bold and shy fish. These results partially support a pace-of-life syndrome linking boldness and aerobic metabolism in juvenile bluegill sunfish, but did not reveal a link between boldness and anaerobic metabolism. Our findings suggest that aerobic and anaerobic capacities may be subject to different selection pressures, and that physiological processes governing maximum anaerobic performance in fishes are independent from behavioural and physiological traits related to boldness