Single-molecule Measurements of DNA Topology and Topoisomerases

Topological properties of DNA influence its mechanical and biochemical interactions. Genomic DNA is maintained in a state of topological homeostasis by topoisomerases and is subjected to mechanical stress arising from replication and segregation. Despite their fundamental roles, the effects of topology and force have been difficult to ascertain. Developments in single-molecule manipulation techniques have enabled precise control and measurement of the topology of individual DNA molecules under tension. This minireview provides an overview of these single-molecule techniques and illustrates their unique capabilities through a number of specific examples of single-molecule measurements of DNA topology and topoisomerase activity.     

Interfering effects of multitasking on muscle activity in the upper extremity.

Multitasking, where workers are required to perform multiple physical tasks with various levels of cognitive load is common in today's workplace. Simultaneous physical and mental demands are thought to cause task interference and likely increase muscle activity. To test the interfering effects of multitasking, 16 healthy participants performed hand and shoulder exertions with combinations of four grip conditions (no grip, 30% grip with low precision, 30% grip with high precision, and maximal grip) and three shoulder conditions at 90 degrees abduction (maintaining posture, 40% force-controlled moment, 40% posture-controlled moment), with and without the Stroop test while surface EMG was recorded from eight upper extremity muscles. Both 40% MVC shoulder moments increased extrinsic forearm muscle activity by 2-4% MVE (p<0.01). Grip exertion at 30% MVC reduced anterior and middle deltoid activity by 2% MVE (p<0.01). Exerting a constant force against the transducer (force-controlled) required 3-4% MVE greater middle and posterior deltoid activity (p<0.001) compared to supporting an equivalent inertial load at the same shoulder angle (posture-controlled). Performing the mental task (Stroop test) concurrently with either 40% MVC shoulder moments significantly increased trapezius activity by nearly 2% MVE (p<0.05). Interestingly, the Stroop test also reduced all deltoid activity by 1% MVE (p<0.05). The addition of both the Stroop test and force-control shoulder exertion independently reduced maximal grip force by 7% and 10% MVC, respectively. These results suggest that more complex workplace tasks may act to increase muscle load or interfere with task performance. These small but significant findings may play a role in the development of long-term musculoskeletal disorders in the workplace.     

Multiple duplications of yeast hexose transport genes in response to selection in a glucose-limited environment

When microbes evolve in a continuous, nutrient-limited environment, natural selection can be predicted to favor genetic changes that give cells greater access to limiting substrate. We analyzed a population of baker's yeast that underwent 450 generations of glucose-limited growth. Relative to the strain used as the inoculum, the predominant cell type at the end of this experiment sustains growth at significantly lower steady-state glucose concentrations and demonstrates markedly enhanced cell yield per mole glucose, significantly enhanced high-affinity glucose transport, and greater relative fitness in pairwise competition. These changes are correlated with increased levels of mRNA hybridizing to probe generated from the hexose transport locus HXT6. Further analysis of the evolved strain reveals the existence of multiple tandem duplications involving two highly similar, high- affinity hexose transport loci, HXT6 and HXT7. Selection appears to have favored changes that result in the formation of more than three chimeric genes derived from the upstream promoter of the HXT7 gene and the coding sequence of HXT6. We propose a genetic mechanism to account for these changes and speculate as to their adaptive significance in the context of gene duplication as a common response of microorganisms to nutrient limitation.      

Differential susceptibility of subsarcolemmal and intermyofibrillar mitochondria to apoptotic stimuli

Apoptosis can be evoked by reactive oxygen species (ROS)-induced mitochondrial release of the proapoptotic factors cytochrome c and apoptosis-inducing factor (AIF). Because skeletal muscle is composed of two mitochondrial subfractions that reside in distinct subcellular regions, we investigated the apoptotic susceptibility of subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria. SS and IMF mitochondria exhibited a dose-dependent release of protein in response to H₂O₂ (0, 25, 50, and 100 µM). However, IMF mitochondria were more sensitive to H₂O₂ and released a 2.5-fold and 10-fold greater amount of cytochrome c and AIF, respectively, compared with SS mitochondria. This finding coincided with a 44% (P < 0.05) greater rate of opening (maximum rate of absorbance decrease, V_max) of the protein release channel, the mitochondrial permeability transition pore (mtPTP), in IMF mitochondria. IMF mitochondria also exhibited a 47% (P < 0.05) and 60% (0.05 < P < 0.1) greater expression of the key mtPTP component voltage-dependent anion channel and cyclophilin D, respectively, along with a threefold greater cytochrome c content, but similar levels of AIF compared with SS mitochondria. Despite a lower susceptibility to H₂O₂-induced release, SS mitochondria possessed a 10-fold greater Bax-to-Bcl-2 ratio (P < 0.05), a 2.7-fold greater rate of ROS production, and an approximately twofold greater membrane potential compared with IMF mitochondria. The expression of the antioxidant enzyme Mn²⁺-superoxide dismutase was similar between subfractions. Thus the divergent protein composition and function of the mtPTP between SS and IMF mitochondria contributes to a differential release of cytochrome c and AIF in response to ROS. Given the relatively high proportion of IMF mitochondria within a muscle fiber, this subfraction is likely most important in inducing apoptosis when presented with apoptotic stimuli, ultimately leading to myonuclear decay and muscle fiber atrophy. reactive oxygen species; skeletal muscle; mitochondrial permeability transition pore; cytochrome c; apoptosis     

Identification of protein bands in polyacrylamide gel by protein printing

Previously described methods for identification of proteins separated in cylindrical polyacrylamide gels have been found to be costly in time and antiserum and difficult to apply to small amounts of protein as are found in cerebrospinal fluid. We describe a method which involves printing of the proteins on the cut surface of the gel onto nitrocellulose paper. The protein bands of the imprint can then be identified using labelled antibodies. We have found this to be economical and quick, and it has permitted sensitive and reliable identification of proteins in unconcentrated cerebrospinal fluid and aqueous humour.     

A review of computational fluid dynamics applications in pressure-driven membrane filtration

Ongoing advances in computational performance and numerics have led to computational fluid dynamics (CFD) becoming a ubiquitous modelling tool. However, CFD methods have only been adopted to simulate pressure-driven membrane filtration systems relatively recently. This paper reviews various approaches to describing the behaviour of these systems using CFD, beginning with the hydrodynamics of membrane channels, including discussion of laminar, turbulent, and transition flow regimes, with reference to the effects of osmotic pressure, concentration polarisation, and cake formation. The use of CFD in describing mass transfer through the membrane itself is then discussed, followed by some concluding comments on commercial membrane simulation packages and future research directions in membrane CFD.     

Do force-time and power-time measures in a loaded jump squat differentiate between speed performance and playing level in elite and elite junior rugby union players?

The purpose of this study was to investigate the discriminative ability of rebound jump squat force-time and power-time measures in differentiating speed performance and competition level in elite and elite junior rugby union players. Forty professional rugby union players performed 3 rebound jump squats with an external load of 40 kg from which a number of force-time and power-time variables were acquired and analyzed. Additionally, players performed 3 sprints over 30 m with timing gates at 5, 10, and 30 m. Significant differences (p < 0.05) between the fastest 20 and slowest 20 athletes, and elite (n = 25) and elite junior (n = 15) players in speed and force-time and power-time variables were determined using independent sample t-tests. The fastest and slowest sprinters over 10 m differed in peak power (PP) expressed relative to body weight. Over 30 m, there were significant differences in peak velocity and relative PP and rate of power development. There was no significant difference in speed over any distance between elite and elite junior rugby union players; however, a number of force and power variables including peak force, PP, force at 100 milliseconds from minimum force, and force and impulse 200 milliseconds from minimum force were significantly (p < 0.05) different between playing levels. Although only power values expressed relative to body weight were able to differentiate speed performance, both absolute and relative force and power values differentiated playing levels in professional rugby union players. For speed development in rugby union players, training strategies should aim to optimize the athlete's power to weight ratio, and lower body resistance training should focus on movement velocity. For player development to transition elite junior players to elite status, adding lean mass is likely to be most beneficial.     

Reversing the Warburg Effect as a Treatment for Glioblastoma

Glioblastoma multiforme (GBM), like most cancers, possesses a unique bioenergetic state of aerobic glycolysis known as the Warburg effect. Here, we documented that methylene blue (MB) reverses the Warburg effect evidenced by the increasing of oxygen consumption and reduction of lactate production in GBM cell lines. MB decreases GBM cell proliferation and halts the cell cycle in S phase. Through activation of AMP-activated protein kinase, MB inactivates downstream acetyl-CoA carboxylase and decreases cyclin expression. Structure-activity relationship analysis demonstrated that toluidine blue O, an MB derivative with similar bioenergetic actions, exerts similar action in GBM cell proliferation. In contrast, two other MB derivatives, 2-chlorophenothiazine and promethazine, exert no effect on cellular bioenergetics and do not inhibit GBM cell proliferation. MB inhibits cell proliferation in both temozolomide-sensitive and -insensitive GBM cell lines. In a human GBM xenograft model, a single daily dosage of MB does not activate AMP-activated protein kinase signaling, and no tumor regression was observed. In summary, the current study provides the first in vitro proof of concept that reversal of Warburg effect might be a novel therapy for GBM.