A simple protocol for assessing inter-trial and inter-examiner reliability for two noninvasive measures of limb muscle strength.

Noninvasive measures of limb muscle strength are quite useful in preclinical translational studies that use mouse models of muscle disease, peripheral nerve disease, and movement disorders. The present study uses a simple protocol for assessing both inter-trial and inter-examiner reliability for two noninvasive methods of assessing limb strength in dystrophic (mdx) and wild type mice. One method, termed the whole body tension (WBT) method or escape test, measures the total phasic pulling tension exerted by the fore- and hindlimbs while a mouse attempts to escape into a darkened tube. Another procedure, termed the four limb wire grid holding test, measures the minimal amount of sustained tension (physical impulse) exerted by the fore- and hindlimbs while the mouse hangs suspended in an upside-down position. A comparison of the two methods revealed significant inter-trial and inter-examiner correlations in each procedure, although the WBT procedure consistently produced higher correlations than the four limb wire grid holding test. Inter-trial reliability for each test was higher than inter-examiner reliability, indicating that each longitudinal series of tests is best performed by a single investigator. The holding test also did not consistently detect differences between wild type and mdx populations at ages greater than 4 months. These results demonstrate the utility of a simple protocol for assessing the reliability of noninvasive tests that measure limb strength, and should be useful in comparing different functional measures in a broad range of translational studies.

Targeted chemical wedges reveal the role of allosteric DNA modulation in protein-DNA assembly.

The cooperative assembly of multiprotein complexes results from allosteric modulations of DNA structure as well as direct intermolecular contacts between proteins. Such cooperative binding plays a critical role in imparting exquisite sequence specificity on the homeobox transcription factor (Hox) family of developmental transcription factors. A well-characterized example includes the interaction of Hox proteins with extradenticle (Exd), a highly conserved DNA binding transcription factor. Although direct interactions are important, the contribution of indirect interactions toward cooperative assembly of Hox and Exd remains unresolved. Here we use minor groove binding polyamides as structural wedges to induce perturbations at specific base steps within the Exd binding site. We find that allosteric modulation of DNA structure contributes nearly 1.5 kcal/mol to the binding of Exd to DNA, even in the absence of direct Hox contacts. In contrast to previous studies, the sequence-targeted chemical wedges reveal the role of DNA geometry in cooperative assembly of Hox-Exd complexes. Programmable polyamides may well serve as general probes to investigate the role of DNA modulation in the cooperative and highly specific assembly of other protein-DNA complexes.