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Summary Mechanical impact stress on the vocal fold surface, particularly when excessive, has been postulated to cause the so-called phonotraumatic tissue lesions, such as nodules and polyps. The collision stress between the vocal folds depends on the vocal fold velocity at the time of impact. Hence this vocal fold collision speed is a relevant parameter when considering biomechanical economy of phonation, especially in voice professionals needing a louder voice than normal. Combining a precise photometric measurement of glottal area and simultaneous measurements of translaryngeal impedance (electroglottogram) for identifying the time of the maximum rate of increase of vocal fold contact allows computing the vocal fold collision speed in a wide range of loudnesses. The vocal fold collision speed is - for modal voicing - always smaller than the maximum vocal fold velocity during the closing phase, but it strongly increases with intensity. Moreover, this increase shows a biphasic pattern, with a significant enhancement from a certain value of dB on. Understanding physiological variables that influence vocal fold collision forces provides relevant insight into the pathophysiology and the prevention of voice disorders associated with phonotraumatic vocal hyperfunction.

Osteogenesis imperfecta (OI) is a genetic disorder of connective tissue characterized by spontaneous fractures, bone deformities, impaired growth and posture, as well as extra-skeletal manifestations. Recent studies have underlined an impairment of the osteotendinous complex in mice models of OI. The first objective of the present work was to further investigate the properties of tendons in the osteogenesis imperfecta mouse (oim), a model characterized by a mutation in the COL1A2 gene. The second objective was to identify the possible beneficial effects of zoledronic acid on tendons. Oim received a single intravenous injection of zoledronic acid (ZA group) at 5 weeks and were euthanized at 14 weeks. Their tendons were compared with those of untreated oim (oim group) and control mice (WT group) by histology, mechanical tests, western blotting and Raman spectroscopy. The ulnar epiphysis had a significantly lower relative bone surface (BV/TV) in oim than WT mice. The tendon of the triceps brachii was also significantly less birefringent and displayed numerous chondrocytes aligned along the fibers. ZA mice showed an increase in BV/TV of the ulnar epiphysis and in tendon birefringence. The tendon of the flexor digitorum longus was significantly less viscous in oim than WT mice; in ZA-treated mice, there was an improvement of viscoelastic properties, especially in the toe region of stress-strain curve, which corresponds to collagen crimp. The tendons of both oim and ZA groups did not show any significant change in the expression of decorin or tenomodulin. Finally, Raman spectroscopy highlighted differences in material properties between ZA and WT tendons. There was also a significant increase in the rate of hydroxyproline in the tendons of ZA mice compared with oim ones. This study highlighted changes in matrix organization and an alteration of mechanical properties in oim tendons; zoledronic acid treatment had beneficial effects on these parameters. In the future, it will be interesting to better understand the underlying mechanisms which are possibly linked to a greater solicitation of the musculoskeletal system.

Vocal onset is the process occurring between the first detectable oscillatory glottal movement and the steady state vibration of the vocal folds. To some extent, the voice onset mirrors the voice offset. High speed imaging, photo-, electro-, flow- and ultrasonoglottography and sound analysis have been used in combinations to allow detailed qualitative insight into the phenomenon. Moreover, the instantaneous intraglottal pressure can be computed from the combined records of transglottal airflow and glottal area. A large number of vocal onsets of different types were analysed in various conditions of modal healthy phonation. Vocal folds (VF) vibration can start either from a closed glottis (hard onset) or from an open glottis (soft/breathy onset). In a soft onset, the amplitude of oscillations progressively increases over 2 to more than 30 cycles, before the first clear closed plateau is achieved. It is not possible to define whether the first movement of VF is towards medial or lateral. Hard, soft and breathy onsets can be clearly identified. Flow- and photoglottography are the most sensitive signals in detecting the first glottal movements in soft and breathy onsets. The shape of the EGG signal depends on the contact of the VF edges. The duration of the onset phase is to some extent related to VF adduction speed and peak expiratory flow. The ultrasound technique is sensitive, but lacks physiological interpretation. From the first onset-cycles on, the intraglottal pressure during the opening phase of the glottis exceeds that during the closing phase. During soft/breathy onsets with a sufficiently large number of cycles, when the vibrating mass increases, a trend appears toward a slight progressive decrease of the fundamental frequency of the oscillations, likely related to the increasing vibrating mass.

Summary Purpose Smartphone technology provides new opportunities for recording standardized voice samples of patients and transmitting the audio files to the voice laboratory. This drastically improves the achievement of baseline designs, used in research on efficiency of voice treatments. However, the basic requirement is the suitability of smartphones for recording and digitizing pathologic voices (mainly characterized by period perturbations and noise) without significant distortion. In a previous article, this was tested using realistic synthesized deviant voice samples (/a:/) with three precisely known levels of jitter and of noise in all combinations. High correlations were found between jitter and noise to harmonics ratio measured in (1) recordings via smartphones, (2) direct microphone recordings, and (3) sound files generated by the synthesizer. In the present work, similar experiments were performed (1) in the presence of increasing levels of ambient noise and (2) using synthetic deviant voice samples (/a:/) as well as synthetic voice material simulating a deviant short voiced utterance (/aiuaiuaiu/). Results Ambient noise levels up to 50 dB A are acceptable. However, signal processing occurs in some smartphones, and this significantly affects estimates of jitter and noise to harmonics ratio when formant changes are introduced in analogy with running speech. The conclusion is that voice material must provisionally be limited to a sustained /a/.

Vocal folds show a damped oscillatory movement while abducting at the end of a vocal emission. The phenomenon can be observed with high-speed videoendoscopy and with different glottographic methods. It reflects important mechanical properties of the vocal oscillator, and cannot be voluntarily controlled. It could become a valuable clinical parameter, particularly in a medicolegal context, but its large variability in a same subject limits its use. First, possibilities and limitations of each recording method are reviewed. Second, the three main physiological factors accounting for the variability are analysed: (1) the timing dynamics of the expiratory pressure with respect to the opening of the glottis; (2) the speed at which vocal fold edges are abducted and glottal resistance drops, the combined effect of (1) and (2) determining the persisting transglottal flow, hence a persisting driving force; (3) the morphological change of the oscillator, whose lip-like shape becomes flattened depending on the degree of abduction. For clinical/medicolegal applications, additional research is required as to the recording protocol. A possible solution could be an entire recording with high speed transnasal videokymography of a standardised passage read by the subject, with a posteriori automatic extraction, by dedicated software, of all damping phases and computation of the average damping coefficient.

Summary The most frequently observed type of voice onset in spontaneous speech in normal subjects is the soft onset, and it may be considered as the “physiological” onset. It starts from an immobile narrow glottal slit crossed by a continuous airflow, and then a few oscillations (even a single one in some cases) precede the first glottal closure. It is a transient event, during which the acting forces, lung pressure, intraglottal pressure, myoelastic tension of the vocal fold (VF) oscillator and inertance of the supraglottal vocal tract, interact to progressively reach the steady state of a sustained oscillation. Combined measurements of flow, area, and pressure provide a detailed qualitative and quantitative analysis of the intraglottal mechanical events at the precise moment of starting oscillation in a physiological (soft or soft/breathy) onset. Our in vivo measurements of airflow and glottal area show that the very first oscillation occurs exactly at the time when turbulence appears at the level of the glottal narrowing, ie, when the Reynolds number reaches its critical value. The turbulence may be assumed to trigger an oscillator consisting in the ensemble of the VFs and the air of the vocal tract, which is known to be weakly damped. Turbulence can act here as an aspecific flick, triggering the oscillator, the frequency of oscillation being determined by its mechanical properties. Furthermore, the first noticeable glottal oscillations are sinusoidal: the VFs are neither steeply sucked together by a negative Bernoulli pressure, nor burst apart by the lung pressure. Our measurements show that, at the critical time, the rising positive lung pressure is balanced by the rising negative Bernoulli pressure generated by the transglottal flow.

Intraglottal pressure is the driving force of vocal fold vibration. Its time course during the open phase of the vibratory cycle is essential in the mechanics of phonation, but measuring it directly is difficult and may hinder spontaneous voicing. However, it can be computed from the in vivo measured transglottal flow and glottal area (hence the air particle velocity) on the basis of the Bernoulli energy law and the interaction with the inertance of the vocal tract. As to sustained modal phonation, calculations are presented for the two possible shapes of glottal duct: convergent and divergent, including absolute calibration in order to obtain quantitative physical values. Whatever the glottal duct configuration, the calculations based on measured values of glottal area and air flow show that the integrated intraglottal pressure during the opening phase systematically exceeds that during the closing phase, which is the basic condition for sustaining vocal fold oscillation. The key point is that the airflow curve is skewed to the right relative to the glottal area curve. The skewing results from air compressibility and vocal tract inertance. The intraglottal pressure becomes negative during the closing phase. As to the soft (or physiological) voice onset, a similar approach shows that the integrated pressure differences (opening phase − closing phase) actually increase as the onset progresses, and this applies to the results based on Bernoulli's energy law as well as to those based on the interaction with the inertance of the vocal tract. Furthermore and similarly, the phase lead of the pressure wave with respect to the glottal opening progressively increases. The underlying explanation lies in the progressively increasing skewing of the airflow curve to the right with respect to the glottal area curve.

Smartphone technology provides new opportunities for recording standardized voice samples of patients and sending the files by e-mail to the voice laboratory. This drastically improves the collection of baseline data, as used in research on efficiency of voice treatments. However, the basic requirement is the suitability of smartphones for recording and digitizing pathologic voices (mainly characterized by period perturbations and noise) without significant distortion. In this experiment, two smartphones (a very inexpensive one and a high-level one) were tested and compared with direct microphone recordings in a soundproof room. The voice stimuli consisted in synthesized deviant voice samples (median of fundamental frequency: 120 and 200 Hz) with three levels of jitter and three levels of added noise. All voice samples were analyzed using PRAAT software. The results show high correlations between jitter, shimmer, and noise-to-harmonics ratio measured on the recordings via both smartphones, the microphone, and measured directly on the sound files from the synthesizer. Smartphones thus appear adequate for reliable recording and digitizing of pathologic voices.

This article presents a novel application of the 'single line scanning' of the vocal fold vibrations (kymography) in singing pedagogy, particularly in a specific technical voice exercise: the 'messa di voce'. It aims at giving the singer relevant and valid short-term feedback. A user-friendly automatic analysis program makes possible a precise, immediate quantification of the essential physiological parameters characterizing the changes in glottal impedance, concomitant with the progressive increase and decrease of the lung pressure. The data provided by the program show a strong correlation with the hand-made measurements. Additional measurements such as subglottic pressure and flow glottography by inverse filtering can be meaningfully correlated with the data obtained from the kymographic images.

Intraglottal pressure is the driving force of vocal fold vibration. Theoretically, simultaneous quantification of glottal area and transglottal airflow allows the calculation of the intraglottal pressure waveform during a single vibration cycle. In this study, we show that, by combining photoglottography (transglottal light transmission) and airflow (Rothenberg mask) measurements during sustained vocal emissions in vivo, the intraglottal pressure wave can be approximated in a way similar to what has been done in models. The results confirm in vivo that the intraglottal pressure is systematically larger during the opening phase than during the closing phase, so that over one whole cycle, the driving force performs net positive work, accounting for sustained vocal fold motion. A component of this driving force asymmetry is related to vocal tract inertance, which also accounts for the skewing of the airflow waveform compared with the area waveform. Furthermore, the intraglottal pressure ratio (opening:closing) increases with voicing intensity, reaches a maximum around 76 dB, and significantly decreases at higher intensities. This rise and fall suggests that there is a range of intensity values in which, mechanically, a maximum of the driving force is imparted to the vocal fold mass. This finding could have implications for voice economy in professional speakers.

Summary Introduction Inspiratory phonation (IP) means phonating with inspiratory airflow. Some vocalists remarkably master this technique, to such an extent that it offers new dramatic, aesthetic, and functional possibilities in singing specific contemporary music. The present study aims to a better understanding of the physiological backgrounds of IP. Material and methods A total of 51 inhaling utterances were compared with 61 exhaling utterances in a professional soprano highly skilled in inhaling singing, by means of high-speed single-line scanning and advanced acoustic analysis. Ranges of intensity and Fo were kept similar. Results The main differences are: (1) an inversion of the mucosal wave, (2) a smaller closed quotient in IP, (3) a larger opening/closing quotient in IP with the additional difference that in IP, the quotient is larger than 1 (opening slower than closing), whereas it is less than 1 in expiratory mode (opening faster than closing), (4) a larger vocal-fold excursion in IP, (5) higher values of adaptive normalized noise energy in IP, and (6) a steeper slope of harmonic peaks in IP. However, jitter values are similar (within normal range), as well as damping ratios and central formant frequencies. The two voicing modes cannot be differentiated by blind listening. Conclusion The basic physiological mechanisms are comparable in both voicing modes, although with specific differences. IP is actually to be considered as an “extended vocal technique,” a term applied to vocalization in art music, which falls outside of traditional classical singing styles, but with remarkable possibilities in skilled vocalists.

BACKGROUND: We tested the hypothesis that vardenafil, a common drug used for improving erectile dysfunction and able to partially normalize transepithelial chloride transport in cystic fibrosis (CF), modulates CF lung inflammation. METHODS: Inflammatory markers in lungs of F508del-CF and wild-type mice were monitored in response to lipopolysaccharide from Pseudomonas aeruginosa (LPS). The effect of pretreatment with vardenafil (0.14 mg/kg) was evaluated. RESULTS: A latent inflammatory status, characterized by neutrophil infiltrate, mouse macrophage inflammatory protein (MIP)-2 and tumor necrosis factor (TNF)-α, was found in baseline conditions in F508del-CF mice. Inflammatory markers were increased after LPS with higher responses in CF. Vardenafil globally attenuated inflammatory responses in both genotypes however reduction of macrophage infiltration, macrophage chemoattractant chemokine and interleukin-1β was observed in the CF group only. CONCLUSION: Vardenafil reduces lung inflammation with a more pronounced effect in F508del-CF mice, particularly on macrophage cell markers.

Sildenafil and vardenafil, two selective inhibitors of phosphodiesterase type 5 (PDE5) are able, when applied by intraperitoneal injection, to activate chloride transport in cystic fibrosis (CF) mice homozygous for the F508del mutation. Oral treatment with the drugs may be associated with adverse haemodynamic effects. We hypothesised that inhaled PDE5 inhibitors are able to restore ion transport in F508del CF airway epithelium. We developed a restraint-free mouse chamber for inhalation studies. PDE5 inhibitors were nebulised for 15 min at concentrations adjusted from recommended therapeutic oral doses for male erectile dysfunction. We measured in vivo nasal transepithelial potential difference 1 h after a single inhalation of sildenafil, vardenafil or tadalafil in F508del CF and normal homozygous mice. After nebulisation with the drugs in F508del mice, chloride transport, evaluated by perfusing the nasal mucosa with chloride-free buffer containing amiloride followed by forskolin, was normalised; the forskolin response was increased, with the largest values being observed with tadalafil and intermediate values with vardenafil. No detectable effect was observed on sodium conductance. Our results confirm the role of PDE5 inhibitors in restoring chloride transport function of F508del CF transmembrane conductance regulator protein and highlight the potential of inhaled sildenafil, vardenafil and tadalafil as a therapy for CF.

Skeletal muscle contraction is reputed not to depend on extracellular Ca2+. Indeed, stricto sensu , excitation-contraction coupling does not necessitate entry of Ca2+. However, we previously observed that, during sustained activity (repeated contractions), entry of Ca2+is needed to maintain force production. In the present study, we evaluated the possible involvement of the canonical transient receptor potential (TRPC)1 ion channel in this entry of Ca2+and investigated its possible role in muscle function. Patch-clamp experiments reveal the presence of a small-conductance channel (13 pS) that is completely lost in adult fibers from TRPC1−/−mice. The influx of Ca2+through TRPC1 channels represents a minor part of the entry of Ca2+into muscle fibers at rest, and the activity of the channel is not store dependent. The lack of TRPC1 does not affect intracellular Ca2+concentration ([Ca2+]i) transients reached during a single isometric contraction. However, the involvement of TRPC1-related Ca2+entry is clearly emphasized in muscle fatigue. Indeed, muscles from TRPC1−/−mice stimulated repeatedly progressively display lower [Ca2+]itransients than those observed in TRPC1+/+fibers, and they also present an accentuated progressive loss of force. Interestingly, muscles from TRPC1−/−mice display a smaller fiber cross-sectional area, generate less force per cross-sectional area, and contain less myofibrillar proteins than their controls. They do not present other signs of myopathy. In agreement with in vitro experiments, TRPC1−/−mice present an important decrease of endurance of physical activity. We conclude that TRPC1 ion channels modulate the entry of Ca2+during repeated contractions and help muscles to maintain their force during sustained repeated contractions.

An imbalance in (n-6)/(n-3) PUFA has been reported in cystic fibrosis (CF) patients. Glycerophospholipids enriched in docosahexaenoic acid (GPL-DHA) have been shown to regulate the (n-6)/(n-3) fatty acid ratio in the elderly. Here, we tested the effect of GPL-DHA supplementation on PUFA status in F508del homozygous CF mice. GPL-DHA liposomes were administrated by gavage (60 mg DHA/kg daily, i.e. at maximum 1.4 mg DHA/d) to 1.5-mo-old CF mice (CF+DHA) and their corresponding wild-type (WT) homozygous littermates (WT+DHA) for 6 wk. The PUFA status of different tissues was determined by GC and compared with control groups (CF and WT). There was an alteration in the (n-6) PUFA pathway in several CF-target organs in CF compared with WT mice, as evidenced by a higher level of arachidonic acid (AA) in membrane phospholipids or whole tissue (21 and 39% in duodenum-jejunum, 32 and 38% in ileum, and 19 and 43% in pancreas). Elevated AA levels were associated with lower linoleic acid (LA) and higher dihomo-gamma-linolenic acid levels. No DHA deficiency was observed. GPL-DHA treatment resulted in different PUFA composition changes depending on the tissue (increase in LA, decrease in elevated AA, DHA increase, increase in (n-6)/(n-3) fatty acid ratio). However, the DHA/AA ratio consistently increased in all tissues in CF+DHA and WT+DHA mice. Our study demonstrates the effectiveness of an original oral DHA formulation in counter-balancing the abnormal (n-6) fatty acid metabolism in organs of CF mice when administrated at a low dose and highlights the potential of the use of GPL-DHA as nutritherapy for CF patients.

N-butyldeoxynojyrimicin (NB-DNJ, miglustat [Zavesca]) an approved drug for treating Gaucher disease, was reported to be able to correct the defective trafficking of the F508del-CFTR protein.To evaluate the efficacy of in vivo airway delivery of miglustat for restoring ion transport in cystic fibrosis (CF).We used nasal transepithelial potential difference (PD) as a measure of sodium and chloride transport. The effect of nasal instillation of a single dose of miglustat was investigated in F508del, cftr knockout and normal homozygous mice. The galactose iminosugar analog N-butyldeoxygalactonojirimycin (NB-DGJ) was used as a placebo.In F508del mice, sodium conductance (evaluated by basal hyperpolarization) and chloride conductance (evaluated by perfusing the nasal mucosa with chloride-free solution in the presence of amiloride and forskolin) were normalized 1 hour after an intranasal dose of 50 picomoles of miglustat. Chloride conductance in the presence of 200 microM 4-4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS), an inhibitor of alternative chloride channels, was much higher after miglustat than after placebo. In cftr knockout mice, a normalizing effect was observed on sodium but not on chloride conductance.Our results provide clear evidence that nasal delivery of miglustat, at picomolar doses, normalizes sodium and Cftr-dependent chloride transport in F508del transgenic mice; they highlight the potential of topical miglustat as a therapy for CF.

Essential fatty acid deficiency has been increasingly reported in patients with cystic fibrosis. The purpose of this work is to critically summarize previous data on fatty acid status and omega3 supplementation in cystic fibrosis. Although the reported abnormalities differ from study to study, the two most consistent features appeared to be reduced circulating levels of linoleic acid and docosahexaenoic acid (DHA). On the assumption that the fatty acid composition of erythrocyte cell membranes may be similar to that of other organs, it seems appropriate to monitor the phospholipid profile from erythrocyte membranes together with circulating blood levels. Formulations containing widely variable DHA doses, ranging from 300 mg to 5 g per day, have been administered to patients with cystic fibrosis with discrepant outcomes. Randomized controlled trials are needed in order to draw firm conclusions on the therapeutic effect of omega3 fatty acid supplementation in cystic fibrosis.

In skeletal muscle, Ca(2+) is implicated in contraction, and in regulation of gene expression. An alteration of [Ca(2+)](i) homeostasis is responsible, at least partially, for the muscle degeneration that occurs after eccentric contractions in Duchenne muscular dystrophy, a disease characterized by the loss of the cytoskeletal protein dystrophin. Using patch clamp in the cell-attached configuration, we characterized the store-operated channels (SOCs) and the stretch-activated channels (SACs) present in isolated mouse skeletal muscle. SOCs were voltage independent, had a unitary conductance between 7 and 8 pS (110 mm Ca(2+) in the pipette), and their open probability increased when the sarcoplasmic reticulum was depleted by thapsigargin. These SOCs were identical to those previously described in the pathophysiology of Duchenne muscular dystrophy. Under the same experimental conditions, we detected a channel activity that was increased by applying a negative pressure to the patch electrode. The SACs responsible for this current had the same unitary conductance and current-voltage relationship as those observed for SOCs. SOCs and SACs had a similar sensitivity to pharmacological agents such as Gd(3+), SKF-96365, 2-aminoethoxydiphenyl borate and GsMTx4 toxin. Moreover, stimulation with IGF-1 increased the occurrence of the activity of both channel types. Together, these observations suggest that SOCs and SACs might belong to the same population or share common constituents. From a functional point of view, treatment of soleus muscle with SKF-96365 or GsMTx4 toxin increased its sensitivity to a fatigue protocol, suggesting that the influx of Ca(2+) that occurs through these channels during contraction is also involved in force maintaining during repeated stimulations.

Plasticity of voice quality is defined here as the degree of improvement in deviant voice quality that can be achieved immediately or quasi-immediately by changing basic voicing conditions, posture, articulation or resonance, breathing mechanics, laryngeal position, or auditory feedback. Thirty-two adult patients with various benign organic voice pathologies, and who had a (preoperative) functional voice therapy, were scored before therapy using a weighted multidimensional Index of Voice Plasticity (IVP). The hypothesis is that IVP could be a predictor of the final outcome of functional voice therapy, and therefore a correlation with a comparable quantification of the actual results of the therapy was investigated. The IVP shows a satisfactory correlation (Spearman's rho = 0.68) with the efficacy of (preoperative) voice therapy. The IVP also significantly differs between diagnostic categories. Although its predictive value remains limited, the Index of Voice Plasticity seems helpful in decision making for indication of (presurgical) voice therapy.

In earlier work we showed that low relative humidity (RH) of inhaled air causes acoustic voice parameters such as jitter and shimmer to deteriorate. Other authors have shown negative effects on vocal efficiency. To explain these changes in the mechanical properties of the vocal folds, the effects of changes in RH of the air passing over microdissected mucosa of sheep larynges were studied. The dissected surface of the tissue specimen just touched Ringer solution and air of varying RH was blown over the specimen. The mucosa specimen was subjected to sinusoidal oscillations of length (strain) and the resulting force (stress) was measured. The gain and phase angle between the imposed strain and resulting stress were measured, and elasticity and viscosity were calculated. Two different air conditions were tested: air with high RH (100%) vs air with low RH (0%). Viscosity and stiffness increased significantly in both ambient conditions (P < 0.01). Dry dehydrating air resulted in a stiffer and more viscous cover than humid air (P < 0.001). These changes in mechanical characteristics may contribute to the effects on voice parameters described in earlier work.

We have used a new approach to study the effects of overload on skeletal muscle phenotype in mice. The method used avoids any traumatising contact with muscles and the inflammatory reaction that this may provoke. Blocks of lead embedded in silicone were inserted under the skin of the lower part of the back. After 1 month, a 17% hypertrophy was found to have occurred in the tonic soleus muscle, but no change was observed in the fast-twitch extensor digitorum longus (EDL) muscle. The main effects on the contractile properties of the soleus muscle were a decrease in the tetanic relaxation rate and a reduction in the maximal velocity of shortening. Immunohistological analysis of the soleus muscles revealed an increase in the proportion of fibres that express myosin heavy chain (MHC) 1, from 54.2% to 73.9%, with a reduction in the proportion of MHC2a-positive fibres, from 45.8% to 30.2%. These changes were accompanied by an increase in the proportion of fibres that express the slow type of sarcoplasmic reticulum calcium pump (SERCA2a), from 61.8% to 84.7%. In EDL muscles, overload induced only minor changes. Thus, this method of overload affected the soleus muscle in particular. The observed changes in the control of muscle contraction were significantly larger than the changes in typical myofibrillar properties that were observed. These results indicate that there is a temporal dissociation between the relative expression of MHCs and SERCAs.

Sustained phonations were compared in two groups of children (aged 7-12), one with special artistic voice education and one from a normal school, without voice complaints or problems. The hypothesis of specific (better) biomechanical vocal fold properties in the first group is confronted with the hypothesis of differences solely related to training of voice control. In both groups, Fo-aperiodicity was measured in a sustained phonation at 3 different SPL levels. As a general rule, aperiodicity clearly decreases when the voice becomes louder. Aperiodicity is highly significantly lower, at all SPL-levels, in children with trained singing voices: this implies better mechanical properties of the vocal oscillator. The Fo/SPL relation on a sustained /a:/ does not differ in trained and untrained children's voices: out of singing context, trained children do not spontaneously control the Fo/SPL dynamics differently from untrained children. The higher regularity of vocal fold pulses is not related to the duration of training.

Owing to an increasing number of requests for compensation, a medicolegal decision-making system for tinnitus related to noise-induced hearing loss (NIHL) has been elaborated at the Federal Belgian Institute of Occupational Diseases. Experience with 113 patients, all of them claiming compensation for NIHL and tinnitus, is now available. The patients underwent an exhaustive audiological investigation, and their professional career and noise exposure were carefully and objectively documented. We reviewed the group of 35 "accepted" cases (i.e., with chronic tinnitus recognized as related to NIHL and financially compensated as an occupational disease) and analyzed the medicolegal arguments for acceptance or rejection. In these patients, tinnitus was mostly bilateral, was perceived on average at a frequency of 4 KHz and with a supraliminal intensity of 7.2 dB, and lasted on average for 7.3 years. To gain better insight into the relationship between cochlear damage and chronic tinnitus, we compared our group to a control group of 35 patients with similar hearing thresholds at 3 and 4 KHz but free of tinnitus. The main difference is a significantly steeper slope of the audiometric curve between 2 and 3 KHz in the tinnitus group. Furthermore, a notch in the distortion product-gram is noticed in 60% of the ears affected by tinnitus versus 9% of the ears in the control group. This abrupt discontinuity in the activity along the tonotopic axis of the auditory system-the main characteristic of NIHL-could be a factor eliciting tinnitus, as a correspondence between the audiometric notch and tinnitus frequency appears to exist.

Duchenne myopathy is a lethal disease due to the absence of dystrophin, a cytoskeletal protein. Muscles from dystrophin-deficient mice (mdx) typically present an exaggerated susceptibility to eccentric work characterized by an important force drop and an increased membrane permeability consecutive to repeated lengthening contractions. The present study shows that mdx muscles are largely protected from eccentric work-induced damage by overexpressing a dominant negative mutant of TRPV2 ion channel. This observation points out the role of TRPV2 channel in the physiopathology of Duchenne muscular dystrophy.

PURPOSE: To study the effect of plyometric training on Ca2+ sensitivity and the influence of troponin T (TnT) isoforms on Ca2+ -activation properties in skinned human muscle fibers. METHODS: Biopsies were obtained from the vastus lateralis of eight men before and after the training period. Chemically skinned fibers were evaluated regarding their Ca2+ -activation properties and were classified according to their myosin heavy chain (MHC) contents and analyzed regarding their slow and fast TnT isoforms. RESULTS: After training, significant improvements (P < 0.05) were found for static jump, countermovement jump, 6 x 5-m shuttle-run test, and leg-press performances. An 8% increase in the proportion of type IIa fibers (P < 0.05) was observed. Single-fiber diameters increased by 11% in type I (P < 0.01), 10% in type IIa (P < 0.001), and 15% in type IIa/IIx fibers (P < 0.001). Peak fiber force increased by 35% in type I (P < 0.001), 25% in type IIa (P < 0.001), and 57% in type IIa/IIx fibers (P < 0.01). The Ca2+ -activation threshold was not altered by training, but the Ca2+ concentration required to elicit half-maximal activation showed a decreasing trend, with significant changes in type I fibers (P < 0.001). Cooperativity at low Ca2+ concentrations was increased in type I and type IIa/IIx fibers (P < 0.05). Type I fibers exclusively expressed slow TnT isoforms, and type II fibers were always associated with fast TnT isoforms, independent of training status. Therefore, changes in Ca2+ sensitivity after training could not be explained by differential fast or slow TnT isoform expression. CONCLUSION: Plyometric training increased single-fiber Ca2+ sensitivity, especially in type I fibers. These changes could not be explained by a modified TnT isoform expression pattern.

Background Inflammation plays a critical role in lung disease development and progression in cystic fibrosis. Azithromycin is used for the treatment of cystic fibrosis lung disease, although its mechanisms of action are poorly understood. We tested the hypothesis that azithromycin modulates lung inflammation in cystic fibrosis mice. Methods We monitored cellular and molecular inflammatory markers in lungs of cystic fibrosis mutant mice homozygous for the ΔF508 mutation and their littermate controls, either in baseline conditions or after induction of acute inflammation by intratracheal instillation of lipopolysaccharide from Pseudomonas aeruginosa, which would be independent of interactions of bacteria with epithelial cells. The effect of azithromycin pretreatment (10 mg/kg/day) given by oral administration for 4 weeks was evaluated. Results In naive cystic fibrosis mice, a spontaneous lung inflammation was observed, characterized by macrophage and neutrophil infiltration, and increased intra-luminal content of the pro-inflammatory cytokine macrophage inflammatory protein-2. After induced inflammation, cystic fibrosis mice combined exaggerated cellular infiltration and lower anti-inflammatory interleukin-10 production. In cystic fibrosis mice, azithromycin attenuated cellular infiltration in both baseline and induced inflammatory condition, and inhibited cytokine (tumor necrosis factor-α and macrophage inflammatory protein-2) release in lipopolysaccharide-induced inflammation. Conclusion Our findings further support the concept that inflammatory responses are upregulated in cystic fibrosis. Azithromycin reduces some lung inflammation outcome measures in cystic fibrosis mice. We postulate that some of the benefits of azithromycin treatment in cystic fibrosis patients are due to modulation of lung inflammation.

Numerous difficulties arise during in vivo measurements of transepithelial nasal potential difference (PD) in mice, such as inadequate duration and depth of anaesthesia, bronchoaspiration of solutions perfused in the nose, and respiratory and/or cardiovascular depression. Anaesthesia was induced in adult C57 mice with intraperitoneal injection of a combination of fentanyl, droperidol and medetomidine, each of these at either a small dose (0.20, 10 and 0.33 mg/kg, respectively) or at a large dose (0.40, 20 and 0.40 mg/kg, respectively), combined with a fixed dose of 0.375 μg clonidine. In order to establish a pharmacokinetic–pharmacodynamic relationship, blood concentrations of the first three drugs were measured in 24 animals by liquid-chromatography tandem mass spectrometry. At the end of the experiment, naloxone, a competitive morphinic antagonist, and atipamezole, an α-2 adrenergic antagonist, were administered. Bronchoaspiration was prevented by tilting the animal head downwards and by absorbing the excess fluid from the opposite nostril and from the oral cavity. Optimal assessment of anaesthesia associated with regular respiration, loss of blink, pupillary and pedal withdrawal reflexes was obtained with doses of fentanyl, droperidol and medetomidine corresponding to 0.20, 20 and 0.40 mg/kg, respectively. Blood concentrations of fentanyl around 17 ng/mL induced loss of respiratory efforts and were followed by death during the experiment. Integrity of ion transport was demonstrated under continuous perfusion by successive depolarization after amiloride and repolarization after chloride-free solution. The combination investigated in this study lead to adequate surgical anaesthesia (stage III, plane 2) for prolonged nasal PD measurements in spontaneously breathing mice.

In some patients with occupational noise-induced hearing loss, a significant aspect of the handicap concerns the concomitant tinnitus; thus, this disorder must be considered in evaluating a disability percentage in the insurance context. The main difficulty comes from the lack of objective measures for tinnitus. To reach a maximum of medicolegal objectivity, a system was developed within the Belgian Institute of Occupational Disorders (Brussels) in the form of a four-level decision structure, after exhaustive but noninvasive assessment of patients. An aggregate of multiple-choice responses (affirmative, neutral, negative) to elementary questions leads to a decision of the next level, which in turn determines--together with the other decisions at the same level--the conclusion at a still higher level. A positive outcome on all four level-3 questions is required for recognition of noise-induced hearing loss-related tinnitus as an occupational disorder and for financial compensation (final decision, level 4). We assessed 10 exemplary files on which this system was applied by four experts rating independently. A variant of Cohen's Kappa for multiple raters demonstrated high interrater consistency at the first level. In all cases, the decisions at levels 3 and 4 were identical. In this way, the final medicolegal decision relies on standardized criteria and becomes perfectly transparent.

Transepithelial nasal potential difference (NPD) measurements assess ion conductance in the upper respiratory epithelium. NPD is useful in assisting in the diagnosis of classical and atypical cystic fibrosis (CF) and of cystic fibrosis transmembrane regulator (CFTR)-related disorders, as well as for monitoring the effect of pharmacological agents and gene transfer approaches to correct the abnormalities of ion transport in CF. The article summarizes the objectives and the principle of NPD measurements, describes a hands-on protocol of the procedure and provides quality control measures, practical hints and troubleshooting.

The effects of muscle creatine manipulation on contractile properties in oxidative and glycolytic muscles were evaluated. Whereas control mice (NMRi; n = 12) received normal chow (5 g daily), three experimental groups were created by adding creatine monohydrate (CR group; 5%, 1 week; n = 13); beta-guanidinoproprionic acid, an inhibitor of cellular creatine uptake (beta-GPA group; 1%, 2 weeks; n = 12); or CR following beta-GPA (beta-GPA+CR group; n = 11). Total creatine (TCr) and the contractile properties of incubated soleus and extensor digitorum longus (EDL) muscles were determined. For the soleus, compared with control, TCr increased in the CR group (+25%), decreased in beta-GPA group (-50%), and remained stable in the beta-GPA+CR group, whereas, for the EDL, TCr was similar in the CR, and lower in the beta-GPA (-40%) and beta-GPA+CR (-15%) groups. None of the experimental groups (CR, beta-GPA, or beta-GPA+CR) showed changes in peak tension (P(peak)), time to peak tension, or relaxation in soleus or EDL during twitch or tetanic stimulation. For the soleus, fatigue reduced P(peak) to approximately 60% of initial P(peak); 5 min of recovery restored P(peak) to values approximately 15% higher in CR than in controls. P(peak) recovery was not affected by beta-GPA or beta-GPA+CR in the soleus or any treatment in the EDL. Thus, peak tension recovery is enhanced by creatine intake in oxidative but not glycolytic muscles. This may be implicated in the beneficial action of creatine loading.

Dystrophic mice (mdx) and their controls (C57/Bl10) were fed for 1 month with a diet with or without creatine (Cr) enrichment. Cr supplementation reduced mass (by 19%, P < 0.01) and mean fiber surface (by 25%, P < 0.05) of fast-twitch mdx muscles. In both strains, tetanic tension increased slightly (9.2%) without reaching statistical significance (P = 0.08), and relaxation time increased by 16% (P < 0.001). However, Cr had no protective effect on the other hallmarks of dystrophy such as susceptibility to eccentric contractions; large numbers of centrally nucleated fibers in tibialis anterior; and elevated total calcium content, which increased by 85% (P = 0.008) in gastrocnemius mdx muscles. In conclusion, Cr may be a positive intervention for improving function of dystrophic muscle.

Nasal potential difference (NPD) measurements have been proposed to assess defective ion transport in cystic fibrosis (CF). Implementing it routinely is, however, difficult. Therefore, a modified method based on nasal instillation in supine position at reduced flow rate was tested to evaluate its ability to discriminate CF from non-CF subjects. Classical and modified methods were compared in nine healthy subjects and there were no statistical differences. Following the new method, 97 tests were performed on 74 subjects divided in three cohorts: 21 CF patients and two control groups consisting of 19 patients with other pulmonary diseases and 34 healthy subjects. Twenty five children were enrolled in this study. Maximal NPD in CF patients (-44.9 +/- 2.5 mV) was significantly different from that obtained in control groups (-18.1 +/- 1.6 and -17.2 +/- 1.1 mV). Depolarization after amiloride also discriminated CF patients (25.9 +/- 1.4 mV) from control groups (10.5 +/- 0.9 and 8.1 +/- 0.7 mV). Marked repolarization following isoprenaline plus amiloride in low chloride solution was seen in control groups (-15.7 +/- 1.1 and -15.3 +/- 1.1 mV). We conclude that the modified method represents a simplified and equally effective approach to discriminate CF patients from non-CF subjects. Moreover, this method presents practical advantages for the patients related to hygiene and convenience, favoring its application in small children.

Guanidinoethane sulfonic acid (GES), a chemical and biological analog of taurine, decreases rat muscle taurine content when added to drinking water. Over the same period, GES appears in muscle. GES supplementation is often used to study the effect of taurine depletion on physiological mechanisms, without taking into account the possible actions of GES. The purpose of the present study was to investigate the specific actions of GES on contraction of skeletal muscle. In mice EDL muscle, the time delay needed to observe a 20% force decrease after the end of a tetanic stimulation was higher in GES-supplemented than in control muscle. This observation in GES-supplemented muscle could be explained by the action of taurine or GES on several targets, beside others the rate of Ca2+ uptake by sarcoplasmic reticulum (SR) and the Ca2+ sensitivity of myofilaments. SR of rat EDL was isolated by successive centrifugations. The effect of 20 mM taurine or GES on the rate of Ca2+ uptake by SR was measured with the fluorescent Ca2+ indicator fura-2. The results show that the rate of Ca2+ uptake by SR is not modified in the presence of taurine or GES. The Ca2+ sensitivity of myofilaments was studied in chemically skinned fibers in the presence of 20 mM taurine or GES. Both taurine and GES increased the myofilament sensitivity to Ca2+. Thus, the prolonged relaxation time of GES-supplemented muscle can be attributed to an increase in myofilament sensitivity to Ca2+. This higher sensitivity is not due to a decrease in muscle taurine content but rather to an increased GES concentration.

Clenbuterol induces hypertrophy and a slow-to-fast phenotype change in skeletal muscle, but the signaling mechanisms remain unclear. We hypothesized that clenbuterol could act via local expression of insulin-like growth factor I (IGF-I). Administration of clenbuterol to 3-mo-old female Wistar rats resulted in a 10 and 13% increase of soleus muscle mass after 3 and 9 days, respectively, reaching 16% after 4 wk. When associated with triiodothyronine, clenbuterol induced a dramatic slow-to-fast phenotype change. In parallel, clenbuterol administration induced in soleus muscle a fivefold increase in IGF-I mRNA levels associated with an eightfold increase in IGF-binding protein (IGFBP)-4 and a fivefold increase of IGFBP-5 mRNA levels on day 3. This increased IGF-I gene expression was associated with an increase in muscle IGF-I content, already detected on day 1 and persisting until day 5 without increase in serum IGF-I concentrations. These data show that muscle hypertrophy induced by clenbuterol is associated with a local increase in muscle IGF-I content. They suggest that clenbuterol-induced muscle hypertrophy could be mediated by local production of IGF-I.

The cardiac sarco(endo)plasmic reticulum Ca 2+ -ATPase gene ( ATP2A2 ) encodes the following two different protein isoforms: SERCA2a (muscle-specific) and SERCA2b (ubiquitous). We have investigated whether this isoform specificity is required for normal cardiac function. Gene targeting in mice successfully disrupted the splicing mechanism responsible for generating the SERCA2a isoform. Homozygous SERCA2a −/− mice displayed a complete loss of SERCA2a mRNA and protein resulting in a switch to the SERCA2b isoform. The expression of SERCA2b mRNA and protein in hearts of SERCA2a −/− mice corresponded to only 50% of wild-type SERCA2 levels. Cardiac phospholamban mRNA levels were unaltered in SERCA2a −/− mice, but total phospholamban protein levels increased 2-fold. The transgenic phenotype was characterized by a ≈20% increase in embryonic and neonatal mortality (early phenotype), with histopathologic evidence of major cardiac malformations. Adult SERCA2a −/− animals (adult phenotype) showed a reduced spontaneous nocturnal activity and developed a mild compensatory concentric cardiac hypertrophy with impaired cardiac contractility and relaxation, but preserved β-adrenergic response. Ca 2+ uptake levels in SERCA2a −/− cardiac homogenates were reduced by ≈50%. In isolated cells, relaxation and Ca 2+ removal by the SR were significantly reduced. Comparison of our data with those obtained in mice expressing similar cardiac levels of SERCA2a instead of SERCA2b indicate the importance of the muscle-specific SERCA2a isoform for normal cardiac development and for the cardiac contraction-relaxation cycle.

Gene expression of IGF-I, IGFBP-4 and IGFBP-5 was studied in hindhimb skeletal muscle of mice, which were either overloaded or unloaded for 8 days. Overloading induced a 15% hypertrophy in soleus muscle associated with a 60% increase of IGF-I transcript levels and a doubling of IGFBP-4 mRNA levels. IGFBP-5 mRNA levels were decreased to one third of the control value. Changes in IGFBPs mRNA always preceded changes in IGF-I gene expression. Unloading by hindlimb suspension resulted in atrophy of soleus muscle (20%) and phenotype change towards the fast type associated with a transient decrease of IGF-I mRNA (30%) and a sustained increase (×2) of IGFBP-5 transcript. These alterations in IGFBPs expression, in unloaded or overloaded soleus, suggest that they may play a role in skeletal muscle adaptation to changes in loading.

Summary Expression levels of fast-twitch (SERCA1), slow-twitch (SERCA2a) and “housekeeping” (SERCA2 b) isoforms of the sarcoplasmic reticulum Ca 2+ -transport ATPase were monitored during regeneration of rat soleus muscles following necrosis induced by the toxin notexin at the tissue level by Western blot analysis and at the cellular level by immunocytochemical analysis. Due to necrosis, levels of muscle-specific SERCA1 and SERCA2 a isoforms dropped to low levels on the third day after injection of the toxin. Subsequently, during regeneration both isoforms recovered but with a different time course. Expression of the fast type SERCA1 increased first. This type showed its most pronounced increase between day 3 and 10. Expression of the slow type SERCA2 a was biphasic. After an increase to approximately one third of the control value on days 5-10, it showed its main increase up to the control level between day 10 and 21. Expression levels of the housekeeping SERCA2 b isoform remained relatively constant throughout the 4 weeks of regeneration. Between day 10 and 28, when new innervation is established, SERCA2 a expression spread gradually over almost all fibers whereas the number of SERCA1-expressing fibers decreased and only a limited number of fibers co-expressed SERCA1 and SERCA2 a. At 4 weeks of regeneration, expression of the fast isoform was found only in 12% of the fibers, whereas the slow form was found in 98% of the fibers. In the contralateral untreated soleus muscles, 26% SERCA1-positive and 81% SERCA2 a-positive fibers were observed. Immunocytochemical analysis showed that SERCA1 and SERCA2 a were co-expressed with fast and slow myosin isoforms in fibers of normal muscles but in regenerated muscle only slow myosin and slow SERCA isoforms correlated. The results show that during regeneration levels of fast and slow SERCA proteins change in a similar way as their mRNAs do. However, in regenerated soleus, unlike in normal muscle, expression of slow SERCA is coregulated only with the slow myosin isoform. This finding is in agreement with the fact that the number of slow type fibers is increased in regenerated soleus.

Parallel and series compliances have been studied in extensor digitorum longus (fast) and soleus (slow) muscles in normal (strain C57BL/10) and dystrophic (strain mdx) mice, and in muscles regenerated after transplantation. Resting elasticity was analysed by measuring the tension-length relationship in unstimulated muscles. Passive tension is larger in mdx and in transplanted muscles than in their normal controls: at 1.15L0, passive tension is 0.12P0 in controls, 0.32P0 in mdx, and 0.40P0 in transplanted muscles. The differences are statistically significant. This indicates that passive tension is mainly determined by the amount of connective tissue. The stress-strain relationship of the series compliance was analysed by the method of isotonic releases during tetani. It is adequately described by a single exponential equation, with a rate constant of about 8P0/P. No difference was found between the various muscles studied. Extension of the series compliance at P0 is 0.02L0. Compliance is about 3% (L0/P0) at 0.2P0, and is not significantly different from zero at P0. Series compliance is thus very low in mouse muscles, substantially lower than in frog and rat muscles. Consequently, it can be neglected in studies of whole mouse muscle in which tension is higher than 0.4P0.

We report the development of hypothermia following induction chemotherapy in a patient with stage IV Hodgkin's disease, fever, and hypotension. A link between hypothermia, hypotension, and sensory neuropathy is possible. Acute autonomic neuropathy is the suggested cause.