Activation of Soluble Polysaccharides with 1-Cyano-4-Dimethylaminopyridine Tetrafluoroborate (CDAP) for Use in Protein–Polysaccharide Conjugate Vaccines and Immunological Reagents. III Optimization of CDAP Activation

CDAP (1-cyano-4-dimethylaminopyridine tetrafluoroborate) is employed in the synthesis of conjugate vaccines as a cyanylating reagent. In the published method, which used pH 9 activation at 20 °C (Vaccine, 14:190, 1996), the rapid reaction made the process difficult to control. Here, we describe optimizing CDAP activation using dextran as a model polysaccharide. CDAP stability and reactivity were determined as a function of time, pH and temperature. While the rate of dextran activation was slower at lower pH and temperature, it was balanced by the increased stability of CDAP, which left more reagent available for reaction. Whereas maximal activation took less than 2.5 min at pH 9 and 20 °C, it took 10–15 min at 0 °C. At pH 7 and 0 °C, the optimal time increased to >3 h to achieve a high level of activation. Many buffers interfered with CDAP activation, but DMAP could be used to preadjust the pH of polysaccharide solutions so that the pH only needed to be maintained. We found that the stability of the activated dextran was relatively independent of pH over the range of pH 1–9, with the level of activation decreased by 40–60% over 2 h. The use of low temperature and a less basic pH, with an optimum reaction time, requires less CDAP, improving activation levels while making the process more reliable and easier to scale up.

Independent control of cocontraction and reciprocal activity during goal-directed reaching in muscle space

The movement in a joint is facilitated by a pair of muscles that pull in opposite directions. The difference in the pair’s muscle force or reciprocal activity results in joint torque, while the overlapping muscle force or the cocontraction is related to the joint’s stiffness. Cocontraction knowingly adapts implicitly over a number of movements, but it is unclear whether the central nervous system can actively regulate cocontraction in a goal-directed manner in a short span of time. We developed a muscle interface where a cursor’s horizontal position was determined by the reciprocal activity of the shoulder flexion–extension muscle pair, while the vertical position was controlled by its cocontraction. Participants made goal-directed movements to single and via-point targets in the two-dimensional muscle space, learning to move the cursor along the shortest path. Simulations using an optimal control framework suggest that the reciprocal activity and the cocontraction may be controlled independently by the CNS, albeit at a rate orders of magnitude slower than the muscle’s maximal activation speed.

Comparison of muscle activation and barbell kinematics during bench press with different loads

The aim of this study was to compare barbell kinematics and muscle patterning in bench press with different loads, but with maximum effort, in young males with resistance training experience. Ten healthy experiences strength-training males (aged 27.3±5.9 years, body mass 82.8±16.6 kg, height 1.78±0.05 m, experience 7.3±4.2 years) performed maximal effort bench presses (1–2 repetitions) with loads varying from 30%, with 10% increments until 100% of 1-RM. Muscle activity of seven muscles and barbell kinematics were measured during descending and ascending phases. Average and peak upwards lifting velocity increased, while lifting time decreases with each decreasing load. In general, the maximal activation of most muscles decreases with decreasing loads, but it was not linear. No effect of loads was shown for the biceps brachii and posterior deltoid muscles. Based upon these findings, it was concluded that maximal lifting velocity may compensate for increased loads, which may allow resistancetrained males who are both healthy or in rehabilitation to avoid heavy loads but experience similar muscle activation. By decreasing the loads, the mechanical stress decreases and time to recover is reduced. Using lower loads with maximal lifting velocity may allow athletes to increase the total volume without increasing the risk of injuries.

Conformational pathway provides unique sensitivity to a synaptic mGluR

Metabotropic glutamate receptors (mGluRs) are dimeric G-protein–coupled receptors that operate at synapses. Macroscopic and single molecule FRET to monitor structural rearrangements in the ligand binding domain (LBD) of the mGluR7/7 homodimer revealed it to have an apparent affinity ~4000-fold lower than other mGluRs and a maximal activation of only ~10%, seemingly too low for activation at synapses. However, mGluR7 heterodimerizes, and we find it to associate with mGluR2 in the hippocampus. Strikingly, the mGluR2/7 heterodimer has high affinity and efficacy. mGluR2/7 shows cooperativity in which an unliganded subunit greatly enhances activation by agonist bound to its heteromeric partner, and a unique conformational pathway to activation, in which mGluR2/7 partially activates in the Apo state, even when its LBDs are held open by antagonist. High sensitivity and an unusually broad dynamic range should enable mGluR2/7 to respond to both glutamate transients from nearby release and spillover from distant synapses.

Determining the optimal maximal and submaximal voluntary contraction tests for normalizing the erector spinae muscles

Background This study aimed to identify which maximum voluntary isometric contraction (MVIC) and sub-MVIC tests produce the highest activation of the erector spinae muscles and the greatest reduction in inter-individual variability, to put them forward as reference normalization maneuvers for future studies. Methods Erector spinae EMG activity was recorded in 38 healthy women during five submaximal and three maximal exercises. Results None of the three MVIC tests generated the maximal activation level in all the participants. The maximal activation level was achieved in 68.4% of cases with the test performed on the roman chair in the horizontal position (96.3 ± 7.3; p < 0.01). Of the five submaximal maneuvers, the one in the horizontal position on the roman chair produced the highest percentage of activation (61.1 ± 16.7; p < 0.01), and one of the lowest inter-individual variability values in the normalized signal of a trunk flexion-extension task. Conclusions A modified Sorensen MVIC test in a horizontal position on a roman chair and against resistance produced the highest erector spinae activation, but not in 100% of participants, so the execution of several normalization maneuvers with the trunk at different inclinations should be considered to normalize the erector spinae EMG signal. A modified Sorensen test in a horizontal position without resistance is the submaximal maneuver that produces the highest muscle activation and the greatest reduction in inter-individual variability, and could be considered a good reference test for normalization.

Impact of heart rate on cross-bridge cycling kinetics in failing and nonfailing human myocardium

The force-frequency relationship (FFR) is an important regulatory mechanism that increases the force-generating capacity as well as the contraction and relaxation kinetics in human cardiac muscle as the heart rate increases. In human heart failure, the normally positive FFR often becomes flat, or even negative. The rate of cross-bridge cycling, which has been reported to affect cardiac output, could be potentially dysregulated and contribute to blunted or negative FFR in heart failure. We recently developed and herein use a novel method for measuring the rate of tension redevelopment. This method allows us to obtain an index of the rate of cross-bridge cycling in intact contracting cardiac trabeculae at physiological temperature and assess physiological properties of cardiac muscles while preserving posttranslational modifications representative of those that occur in vivo. We observed that trabeculae from failing human hearts indeed exhibit an impaired FFR and a reduced speed of relaxation kinetics. However, stimulation frequencies in the lower spectrum did not majorly affect cross-bridge cycling kinetics in nonfailing and failing trabeculae when assessed at maximal activation. Trabeculae from failing human hearts had slightly slower cross-bridge kinetics at 3 Hz as well as reduced capacity to generate force upon K+ contracture at this frequency. We conclude that cross-bridge kinetics at maximal activation in the prevailing in vivo heart rates are not majorly impacted by frequency and are not majorly impacted by disease. NEW & NOTEWORTHY In this study, we confirm that cardiac relaxation kinetics are impaired in filing human myocardium and that cross-bridge cycling rate at resting heart rates does not contribute to this impaired relaxation. At high heart rates, failing myocardium cross-bridge rates are slower than in nonfailing myocardium.

Functional cross-talk between allosteric effects of activating and inhibiting ligands underlies PKM2 regulation

Several enzymes can simultaneously interact with multiple intracellular metabolites, however, how the allosteric effects of distinct ligands are integrated to coordinately control enzymatic activity remains poorly understood. We addressed this question using, as a model system, the glycolytic enzyme pyruvate kinase M2 (PKM2). We show that the PKM2 activator fructose 1,6-bisphosphate (FBP) alone promotes tetramerisation and increases PKM2 activity, but addition of the inhibitor L-phenylalanine (Phe) prevents maximal activation of FBP-bound PKM2 tetramers. We developed a method, AlloHubMat, that uses eigenvalue decomposition of mutual information derived from molecular dynamics trajectories to identify residues that mediate FBP-induced allostery. Experimental mutagenesis of these residues identified PKM2 variants in which activation by FBP remains intact but cannot be attenuated by Phe. Our findings reveal residues involved in FBP-induced allostery that enable the integration of allosteric input from Phe and provide a paradigm for the coordinate regulation of enzymatic activity by simultaneous allosteric inputs.

Distinct Activities of Gli1 and Gli2 in the Absence of Ift88 and the Primary Cilia

The primary cilia play essential roles in Hh-dependent Gli2 activation and Gli3 proteolytic processing in mammals. However, the roles of the cilia in Gli1 activation remain unresolved due to the loss of Gli1 transcription in cilia mutant embryos, and the inability to address this question by overexpression in cultured cells. Here, we address the roles of the cilia in Gli1 activation by expressing Gli1 from the Gli2 locus in mouse embryos. We find that the maximal activation of Gli1 depends on the cilia, but partial activation of Gli1 by Smo-mediated Hh signaling exists in the absence of the cilia. Combined with reduced Gli3 repressors, this partial activation of Gli1 leads to dorsal expansion of V3 interneuron and motor neuron domains in the absence of the cilia. Moreover, expressing Gli1 from the Gli2 locus in the presence of reduced Sufu has no recognizable impact on neural tube patterning, suggesting an imbalance between the dosages of Gli and Sufu does not explain the extra Gli1 activity. Finally, a non-ciliary Gli2 variant present at a higher level than Gli1 when expressed from the Gli2 locus fails to activate Hh pathway ectopically in the absence of the cilia, suggesting that increased protein level is unlikely the major factor underlying the ectopic activation of Hh signaling by Gli1 in the absence of the cilia.

USING THE PERSONALITY-DEVELOPING POTENTIAL OF THE EDUCATIONAL PROCESS IN THE MAGISTRACY OF THE PREPARATION OF THE MANAGER

The peculiarities of the educational process in the magistracy from the standpoint of a personality approach are considered. The specifics of organization of various forms of educational work as a means of development of professionally significant personal qualities of the future manager, forming in him the ability to constructive interaction in management activity are analyzed.It is proved that the educational process in the magistracy of modern high school, and first of all - in the preparation of skilled management personnel, involves maximal activation of the student as a subject of educational, research, as well as - organizational and pedagogical activity, which enables him to purposefully improve personal and professional qualities , necessary for constructive interaction. Present opportunities of classroom and non-classroom work in shaping the personality of the future manager. It is grounded that the improvement of the teaching process, the approach of the content of teaching to the real needs of students, calls for a more detailed study of the applied forms and methods of a personally oriented educational process in view of the realities of the present.