Elasticity of the human false vocal fold

[[abstract]]Objectives: Very little is known about the elasticity of the human ventricular fold (false vocal fold). To better understand the potential role of the false fold in the fluid dynamics and aeroacoustics of phonation, we made some measurements on the elastic properties of human ventricular fold tissues in vitro. Methods: Uniaxial tensile stress-strain characteristics of 6 male and 6 female false fold specimens were quantified with sinusoidal stretch-release deformation. Midcoronal sections of 3 specimens were examined to quantify the relative densities of collagen, elastin, seromucous glandular tissue, and adipose tissue by digital image analysis. Results: Nonlinear stress-strain curves with hysteresis (viscous energy loss) were observed, with large interindividual differences. A hybrid linear-exponential model was used to determine the elastic modulus (tangent Young's modulus) of the false fold. On average, the male false fold was twice as stiff as the female at a tensile strain of 20% to 30%. Conclusions: This preliminary gender-related difference in elasticity could be attributed to a higher proportion of glandular tissue in the female false fold, due to the lower elastic modulus of glands. The present data allow one to develop a more comprehensive biomechanical model of phonation, for optimizing postoperative voice production following laryngeal reconstruction procedures

Relative contributions of collagen and elastin to elasticity of the vocal fold under tension.

[[abstract]]This study examined the contributions of collagen and elastin to the tensile elastic properties of the vocal fold lamina propria. Uniaxial stress–strain responses of vocal fold cover and vocal ligament specimens from 20 human larynges (12 males, 8 females) were quantified with sinusoidal stretch-release deformation in vitro. Mid-coronal sections of 12 specimens were examined histologically with Masson’s trichrome and elastin van Gieson stain to quantify the relative densities of collagen and elastin fibers. Results showed that significantly higher levels of collagen were found in the male vocal fold than female, for both the cover and the ligament. For male there was a significantly higher level of elastin in the cover than in the ligament. On average, the elastic modulus of the male cover was about twice that of the female at high-tensile strain (35–40%), whereas the male ligament was 3–5 times stiffer than the female in the same range. The ligament was stiffer than the cover for male, but the opposite was observed for female. These findings suggested that collagen and elastin could contribute differentially to elasticity of the cover and the ligament. The data may provide guidance for surgical reconstruction and tissue engineering of different lamina propria layers

Structural and Regulatory Elements of HCV NS5B Polymerase – β-Loop and C-Terminal Tail – Are Required for Activity of Allosteric Thumb Site II Inhibitors

<div><p>Elucidation of the mechanism of action of the HCV NS5B polymerase thumb site II inhibitors has presented a challenge. Current opinion holds that these allosteric inhibitors stabilize the closed, inactive enzyme conformation, but how this inhibition is accomplished mechanistically is not well understood. Here, using a panel of NS5B proteins with mutations in key regulatory motifs of NS5B – the C-terminal tail and β-loop – in conjunction with a diverse set of NS5B allosteric inhibitors, we show that thumb site II inhibitors possess a distinct mechanism of action. A combination of enzyme activity studies and direct binding assays reveals that these inhibitors require both regulatory elements to maintain the polymerase inhibitory activity. Removal of either element has little impact on the binding affinity of thumb site II inhibitors, but significantly reduces their potency. NS5B in complex with a thumb site II inhibitor displays a characteristic melting profile that suggests stabilization not only of the thumb domain but also the whole polymerase. Successive truncations of the C-terminal tail and/or removal of the β-loop lead to progressive destabilization of the protein. Furthermore, the thermal unfolding transitions characteristic for thumb site II inhibitor – NS5B complex are absent in the inhibitor – bound constructs in which interactions between C-terminal tail and β-loop are abolished, pointing to the pivotal role of both regulatory elements in communication between domains. Taken together, a comprehensive picture of inhibition by compounds binding to thumb site II emerges: inhibitor binding provides stabilization of the entire polymerase in an inactive, closed conformation, propagated via coupled interactions between the C-terminal tail and β-loop.</p></div

IC₅₀ values for inhibition of RdRp activity of NS5B by Nuc (3′-d′CTP) and NNIs.

<p>Numbers represent mean and standard deviation of IC₅₀ values determined for each inhibitor against the set of NS5B constructs. Heatmap is colored according to IC₅₀ fold shift relative to Δ21 to show changes in inhibition profile for given NNI across NS5B mutant constructs. Thumb site II inhibitors begin to show significant loss of inhibitory potency as interactions between β-loop and C-terminal residues are disrupted by mutations in the interface (Δ21-AAA, truncations past Δ39 and Δ21-Δ39 mutations). Palm site I inhibitor is affected as well, which is explained by disruption of the inhibitor's interaction with the β-loop and C-terminal residues. Inhibition by thumb site I remains for the most part unaffected by NS5B mutations whereas inhibition by palm site II NNI is reduced due to decrease in binding to NS5B with truncated β-loop and/or C-terminal.</p

HCV NS5B polymerase nonnucleoside inhibitors binding sites and NS5B constructs used in studies.

<p>(<b>A</b>) Thumb site I and thumb site II are located on the thumb domain (green); palm site I and palm site II are at the interface of the three domains, thumb, palm (blue) and fingers (red). GS-9669 inhibitor bound in the thumb site II pocket is shown in stick representation (grey, description of crystal structure of NS5B bound to thumb site II inhibitor GS-9669 will be published elsewhere). The active site is indicated by the cyan circle. The other main structural features shown are the C-terminal tail residues (magenta) which contact the β-loop (yellow). (<b>B</b>) 2D representation of domain structure of polymerase and C-terminal truncation sites Δ21, Δ39, Δ47, Δ55, as well as the β-loop deletion mutant Δ21-Δ8 (deleted residues shown in yellow) and LWF triple A mutant F550A/W551A/L553A. Δ55 is a tag free construct and all others contain C6-His. (<b>C</b>) Location of the mutations relative to the tertiary protein structure. (<b>D</b>) Close-up view of interface between LWF motif (magenta, stick representation) and β-loop (yellow) which is dominated by hydrophobic contacts on the surface of the protein.</p

Equilibrium dissociation constants (K_D) for binding of NNIs to Δ21 and fold-shifts in K_D for association to Δ55 and Δ21-Δ8 determined by SPR.

<p>^a Sensorgrams for the binding of NNIs to Δ21, Δ55 and Δ21-Δ8 along with a table of equilibrium and kinetic parameters of interaction are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084808#pone.0084808.s002" target="_blank">Figure S2</a> and Table S1 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084808#pone.0084808.s006" target="_blank">File S1</a>, respectively.</p><p>^b Fold shift in K_D for association of NNIs to Δ55 and Δ21-Δ8 was calculated relative to the K_D determined for binding towards Δ21.</p><p>^c Where K_D values could not be obtained either due to complex binding kinetics (Palm Site II inhibitor binding to Δ55 and Δ21-Δ8) or super-stoichiometric binding (Palm Site I inhibitor binding to Δ21-Δ8), numerical values have been replaced by n/a (not applicable).</p