Objectives/Hypothesis Our laboratory has developed an in vivo rabbit model to investigate the effects of phonation on expression and turnover of the vocal fold extracellular matrix. As a logical outgrowth of this research to include phonotrauma in the present study, we investigated the hypothesis that an increase in airflow rate delivered to the glottis produces a change in glottal configuration and an increase in mean phonation intensity. Study Design Prospective animal study. Methods Six New Zealand white breeder rabbits weighing 3 to 5 kg were used in this study. A rigid endoscope and camera were used to document glottal configuration. Acoustic signals of modal and raised phonation were recorded and digitized. Two separate one-way repeated measures analysis of variance (ANOVA) tests were used to investigate within subject differences in phonation intensity and fundamental frequency between modal and raised phonation. Results Phonation intensity was 54.19 dB SPL (6.21 standard deviations [SD]) during modal phonation, and 60.31 dB SPL (5.68 SD) during raised phonation. Endoscopic images revealed a convergent glottis, with greater separation of the vocal folds during raised phonation. Results of ANOVA revealed a significant within subjects effect for phonation intensity (P = .011). Pairwise comparisons revealed that phonation intensity increased significantly during raised phonation, compared to modal phonation (P = .008). No differences in mean fundamental frequency were observed between phonation conditions. Conclusions Improved understanding of factors that control phonation output in the in vivo rabbit model will result in improved capabilities to match phonation dose across animals and provide immediate direction to future biochemical studies.

Objectives To investigate the hypothesis that a transient episode of raised intensity phonation causes a significant increase in vocal fold inflammatory messenger RNA (mRNA) expression in-vivo. Study Design Prospective animal study. Setting Laboratory. Subjects and Methods Ten New Zealand white breeder rabbits received 30 minutes of experimentally induced modal or raised intensity phonation, followed by a 30 minute recovery period. A separate group of five rabbits served as sham controls. Real-time PCR was performed to investigate the mRNA expression of Interleukin-1beta (IL-1β), Transforming Growth Factor beta-1 (TGFβ-1), and Cyclooxygenase-2 (COX-2). Separate one-way analysis of variance (ANOVA) tests were used to investigate differences in gene expression across groups, with an appropriate alpha correction of .016 to control for type I error. Significant main effects were further examined using Fisher’s Least Significant Difference. Results ANOVA revealed that there were differences for IL-1β, TGF-β1, and COX-2 between sham-control, modal phonation, and raised intensity phonation (p < .0001). Pairwise comparisons revealed that the expression of IL-1β, COX-2, and TGF-β1 increased significantly during raised intensity phonation, compared to modal phonation and sham-control (p < .0001). Conclusions Results provided support for the hypothesis that a transient episode of raised intensity phonation causes a significant increase in vocal fold inflammatory mRNA expression. Future studies will investigate the signal transduction pathways and mechanisms regulating the vocal fold inflammatory response. The long-term goal of these studies is to advance understanding of the molecular and cellular events underlying phonation-related tissue alterations.

The time course of the response and recovery after acute activity seen in exercise is not well understood. The goal of this work is to address how proteins of the thin filament (actin and its capping protein CapZ) are changed by 1 h of mechanical stimulation and return to baseline over time. Neonatal rat ventricular myocytes in culture were subjected to cyclic 10% strain at 1 Hz for 1 h to mimic increased mechanical loading during exercise. CapZ and actin dynamics were analyzed by fluorescence recovery after photobleaching (FRAP) using CapZβ1-GFP, actin-GFP, or actin-RFP. After cyclic strain, CapZ dynamics increased above resting controls and abated 2-3 h after cessation of the cyclic strain. Similarly, actin dynamics initially increased and abated 1.5-2 h after the end of stimulation. Neurohormonal hypertrophic stimulation by phenylephrine or norepinephrine treatments also elevated actin dynamics but required a much longer time of treatment (24-48 h) to be detectable. The actin capping mechanism was explored by use of expression of CapZβ1 with a COOH-terminal deletion (CapZβ1ΔC). Increased dynamics of actin seen with CapZβ1ΔC was similar to the response to cyclic strain. Thus it is possible that mechanical stimulation alters the dynamics for CapZ capping of the actin filament through the CapZβ1 COOH terminus, known as the β tentacle, thereby remodeling sarcomeres in cardiac myocytes. This adaptive mechanism, which is probably regulating thin-filament addition, declines a few hours after the end of a bout of exercise.

Basic fibroblast growth factor induced the up-regulation of HAS-2, HAS-3, MMP-2, and procollagen type I. Histologically, aged vocal folds treated with bFGF revealed increased deposition of HA as compared to sham-treated vocal folds.

Actinorhizal plants are able to establish a symbiotic relationship with Frankia bacteria leading to the formation of root nodules. The symbiotic interaction starts with the exchange of symbiotic signals in the soil between the plant and the bacteria. This molecular dialog involves signaling molecules that are responsible for the specific recognition of the plant host and its endosymbiont. Here we studied two factors potentially involved in signaling between Frankia casuarinae and its actinorhizal host Casuarina glauca: (1) the Root Hair Deforming Factor (CgRHDF) detected using a test based on the characteristic deformation of C. glauca root hairs inoculated with F. casuarinae and (2) a NIN activating factor (CgNINA) which is able to activate the expression of CgNIN, a symbiotic gene expressed during preinfection stages of root hair development. We showed that CgRHDF and CgNINA corresponded to small thermoresistant molecules. Both factors were also hydrophilic and resistant to a chitinase digestion indicating structural differences from rhizobial Nod factors (NFs) or mycorrhizal Myc-LCOs. We also investigated the presence of CgNINA and CgRHDF in 16 Frankia strains representative of Frankia diversity. High levels of root hair deformation (RHD) and activation of ProCgNIN were detected for Casuarina-infective strains from clade Ic and closely related strains from clade Ia unable to nodulate C. glauca. Lower levels were present for distantly related strains belonging to clade III. No CgRHDF or CgNINA could be detected for Frankia coriariae (Clade II) or for uninfective strains from clade IV.

Frankia sp. strain BMG5.30 was isolated from root nodules of a Coriaria myrtifolia seedling on soil collected in Tunisia and represents the second cluster 2 isolate. Frankia sp. strain BMG5.30 was able to re-infect C. myrtifolia generating root nodules. Here, we report its 5.8-Mbp draft genome sequence with a G + C content of 70.03% and 4509 candidate protein-encoding genes.

Objectives/Hypothesis-We investigated acute changes in extracellular matrix gene expression and histologic changes in the deposition of collagen and hyaluronan (HA) from hepatocyte growth factor (HGF) treatment of the aged rat vocal fold. We hypothesized that: 1) HGF induces matrix metalloproteinase gene expression, which may contribute to the downregulation of collagen; and 2) HGF induces hyaluronan synthase (HAS) gene expression, which may play a role in the upregulation of ECM HA.Study Design-prospective animal study.Methods-Fifteen, 18-month old, Sprague-Dawley rats were involved in this study. For gene expression analyses, ten rats were divided into two groups and received serial injections of sham (saline) or HGF (2ng/µL) and sacrificed 2 weeks after the initial injection to investigate acute changes in extracellular matrix gene expression. A separate group of five animals received the above treatment and were sacrificed 4 weeks after the initial injection to investigate histologic changes in the deposition of collagen and HA.Results-Real-time polymerase chain reaction revealed significantly upregulated MMP-2, -9, and HAS-3 messenger RNA (mRNA) expression and significantly downregulated procollagen type I mRNA expression in the HGF-treatment group, compared to the sham-treatment group. Histologic staining revealed significantly reduced collagen deposition and increased deposition of HA in the HGF-treated vocal fold, compared to the sham-treated vocal fold.Conclusions-HGF induced the upregulation of MMP-2, -9, and HAS-3, and downregulated the expression of procollagen type I. Histologically, aged vocal folds treated with HGF revealed decreased collagen deposition, and increased deposition of HA, compared to sham-treated vocal folds.

The heart is exquisitely sensitive to mechanical stimuli and adapts to increased demands for work by enlarging the cardiomyocytes. In order to determine links between mechano-transduction mechanisms and hypertrophy, neonatal rat ventricular myocytes (NRVM) were subjected to physiologic strain for analysis of the dynamics of the actin capping protein, CapZ, and its post-translational modifications (PTM). CapZ binding rates were assessed after strain by fluorescence recovery after photobleaching (FRAP) of green fluorescent protein (GFP) expressed by a GFP-CapZβ1 adenovirus. To assess the role of the protein kinase C epsilon isoform (PKCε), rest or cyclic strain were combined with specific PKCε activation by constitutively active PKCε, or by inhibition with dominant negative PKCε (dnPKCε) expression. Significant increases of CapZ FRAP kinetics with strain were blunted by dnPKCε, suggesting that PKCε is involved in mechano-transduction signaling. Similar combinations of strain and PKC regulation in NRVMs were studied by PTM profiles of CapZβ1 using quantitative two-dimensional gel electrophoresis. The significantly increased charge on CapZ seen with mechanical strain was reversed by the addition of dnPKCε. Potential clinical relevance was confirmed in vivo by PTMs of CapZ in the failing heart of one-year old transgenic mice over-expressing PKCε. Furthermore, with strain there was significant PKCε translocation to the Z-disc and co-localization with CapZβ1 or α-actinin, which was quantified on confocal images. A hypothetical model is presented proposing that one destination of the mechanotransduction signaling pathways might be for PTMs of CapZ thereby regulating actin capping and filament assembly.