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Objectives This study examined the antifibrotic effect of pirfenidone (PFD), which has received regulatory approval in the United States and Japan for treatment of idiopathic pulmonary fibrosis, on the scarred ferret vocal fold (VF) in vivo. Methods Eight male ferrets were divided into two groups: saline and PFD. All animals underwent unilateral scarring under anesthesia. The right VF was electrocauterized with ablation of the entire lamina propria. PFD (1.0 mg/mL) or saline injections into right-side scarred VFs were performed (under an operating microscope) 4 weeks later. After an additional 4 weeks, the larynges were harvested for histological analysis. Prior to harvesting, the ferrets were re-anesthetized, and the VFs were observed and recorded using a rigid video laryngoscope. We immunohistochemically evaluated the expression of collagen types I and III, alpha-smooth muscle actin (α-SMA), and fibronectin in the entire lamina propria. We compared the affected areas (calculated using ImageJ software) between the treated (right) and untreated (left) sides within the same animals and between groups. Results Collagen type I (P = 0.0021) and α-SMA (P = 0.0021) expression levels were lower in the PFD group, but the collagen type III and fibronectin levels did not differ significantly between the two groups. Conclusion PFD injection into the scarred VF is a potentially promising novel antifibrotic treatment. Level of evidence NA Laryngoscope, 130:726-731, 2020.

Aspergillus oryzae is an important microorganism in the bio- and food industries; therefore, understanding the mechanism underlying its secondary metabolism regulation is important for ensuring its safe use. Here, we screened a novel Zn(II)2-Cys6-type protein-encoding gene, AO090003001186, designated as kpeA (kojic acid production enhancement A), from an A. oryzae disruption mutant library of transcriptional regulators. kpeA is highly conserved among filamentous fungi and encodes a protein with Zn(II)2-Cys6 motif located in the middle of the sequence. Phylogenetic analysis revealed that KpeA was classified into a distal group compared to other fungal Zn(II)2-Cys6-type transcriptional regulators. A Cys to Ala substitution mutant of KpeA showed identical phenotype to the kpeA disruption strain, confirming that KpeA is novel type Zn(II)2-Cys6 binding protein. Colonies of the kpeA disruption strain (ΔkpeA) had longer aerial hyphae and showed decreased conidia production. Microscopic analysis suggested that the reduced vesicle size and conidial head formation in ΔkpeA strain account for the decreased conidia production. Transcriptional levels of brlA and downstream abaA and wetA were decreased in ΔkpeA strain. Moreover, ΔkpeA strain produced 6-fold more kojic acid than the control strains, and the expression of kojR and kojA was increased in ΔkpeA strain. Therefore, KpeA is a novel Zn(II)2-Cys6-type protein likely involved in conidiation and kojic acid production at the transcriptional level.

Objectives: To develop a vocal fold (VF) scarring procedure in the ferret, characterize the scars histologically, and test the injectability of the lamina propria (LP). Secondarily, to compare laryngeal anatomy of the ferret with rat and rabbit. Materials and Methods: The larynges of 18 male ferrets were prepared by unilateral scarring, and normal larynges from 6 female Wistar rats and 5 male albino rabbits were used for comparative purposes. For scarring, the right VF were electrocauterized, ablating the entire LP. Prior to harvesting the larynges at 4 and 16 weeks, each ferret was re-anesthetized, and in 3 animals, India ink was injected into the LPs of both normal and scarred VFs. Results: Laryngoscopic methods and instrumentation for precise visualization, scarring, and injection were developed. The scarred VFs had reduced hyaluronic acid and increased collagen type I, III, and fibronectin compared with normal VFs. The 2 timepoints (4 and 16 weeks) differed significantly only in collagen type III level (levels were higher at 4 weeks). Injected ink migrated from scarred LP to muscle layer just beneath the scarred tissue 3 hours after injection. Conclusion: The ferret is a promising species for creation and experimental treatment of vocal fold scar.

OBJECTIVES/HYPOTHESIS Pirfenidone (PFD) is a strong antifibrotic agent that has been clinically approved in Japan for idiopathic pulmonary fibrosis. We examined the antifibrotic effects of PFD on fibroblasts isolated from scarred vocal folds (VFs) of ferrets in vitro. STUDY DESIGN Prospective animal experiments with controls. METHODS Scar fibroblasts (SFs) were isolated from scarred VFs that had been electrocauterized 2 weeks before harvesting (N = 4). Normal fibroblasts (NFs) were isolated from intact VFs (N = 4). SFs and NFs were incubated in the presence of 10 ng/mL transforming growth factor β1 (TGF-β1), with or without PFD. After the 48-hour incubation, mRNA expression levels of α smooth muscle actin (αSMA), TGF-β1, collagen type I, and hyaluronan synthase 2 (HAS2) were examined by real-time polymerase chain reaction. Immunohistochemistry with anti-αSMA anti-collagen type I and phosphorylated Smad (p-Smad)2/3 antibodies in SFs with or without PFD was performed. SFs and NFs were cultured in collagen gel with or without PFD for 48 hours, and the extent of gel contraction was examined quantitatively. RESULTS PFD treatment significantly (P < .05) decreased mRNA expression of collagen type I, significantly increased mRNA expression of TGF-β1 and HAS2, and significantly suppressed collagen gel contraction. However, it did not have a significant effect on the expression of αSMA. The expression of p-Smad2/3 in the nucleus was faded with PFD, possibly demonstrating the suppression of translocation of p-Smad2/3 from cytoplasm to nucleus with PFD. CONCLUSIONS This is the first report to demonstrate the in vitro antifibrotic effects of PFD on fibroblasts isolated from scarred VFs of ferrets. LEVEL OF EVIDENCE NA. Laryngoscope, 128:E171-E177, 2018.

Aspergillus oryzae is an important microorganism in the bio- and food industries; therefore, understanding the mechanism underlying its secondary metabolism regulation is important for ensuring its safe use. Here, we screened a novel Zn(II)

We report on the photoluminescent (PL) properties of carbon nanodots (CNDs) doped with nitrogen and sulfur obtained by the pyrolytic carbonization of amino acids as precursors. Prepared CNDs exhibit stable PL emission under a wide variety of aqueous conditions. The results also provided the way to tune a PL intensity of CNDs by varying the amount of heteroatoms in precursors with a binary mixture of amino acids. The PL quantum yields of the obtained CNDs were determined to be from 0.079 to 0.571.

Objectives/Hypothesis To examine modulation of M-cadherin, a marker for satellite cells (SCs); and MyoD, which may indicate the myogenic activity following recurrent laryngeal nerve (RLN) denervation and immediate reinnervation; and to elucidate the correlation between their modulations and establishment of neuromuscular junctions (NMJs) in the reinnervated rat thyroarytenoid (TA) muscle. Study Design Quantitative real-time polymerase chain reaction qPCR and histologic assessment of the TA muscle following RLN transection and anastomosis. Methods Rats were divided into three groups: 1) denervation alone (DNV) (n = 60), 2) denervation with anastomosis (ANS) (n = 60), and 3) sham-operated controls (n = 12). Animals were sacrificed at 3 days and 1, 3, and 5 weeks after treatment. TA muscles harvested from 40 animals from each DNV and ANS group; all of sham group were subjected to qPCR for assessment of the modulation of M-cadherin and MyoD; and the remaining larynges of DNV and ANS group were used for histologic analysis. Results The expression levels of messenger RNAs (mRNAs) encoding M-cadherin and MyoD in the TA muscle of the DNV group were significantly higher (P

Pirfenidone (PFD) is a strong antifibrotic agent that has been clinically approved in Japan for idiopathic pulmonary fibrosis. We examined the antifibrotic effects of PFD on fibroblasts isolated from scarred vocal folds (VFs) of ferrets in vitro.Prospective animal experiments with controls.Scar fibroblasts (SFs) were isolated from scarred VFs that had been electrocauterized 2 weeks before harvesting (N = 4). Normal fibroblasts (NFs) were isolated from intact VFs (N = 4). SFs and NFs were incubated in the presence of 10 ng/mL transforming growth factor β1 (TGF-β1), with or without PFD. After the 48-hour incubation, mRNA expression levels of α smooth muscle actin (αSMA), TGF-β1, collagen type I, and hyaluronan synthase 2 (HAS2) were examined by real-time polymerase chain reaction. Immunohistochemistry with anti-αSMA anti-collagen type I and phosphorylated Smad (p-Smad)2/3 antibodies in SFs with or without PFD was performed. SFs and NFs were cultured in collagen gel with or without PFD for 48 hours, and the extent of gel contraction was examined quantitatively.PFD treatment significantly (P.05) decreased mRNA expression of collagen type I, significantly increased mRNA expression of TGF-β1 and HAS2, and significantly suppressed collagen gel contraction. However, it did not have a significant effect on the expression of αSMA. The expression of p-Smad2/3 in the nucleus was faded with PFD, possibly demonstrating the suppression of translocation of p-Smad2/3 from cytoplasm to nucleus with PFD.This is the first report to demonstrate the in vitro antifibrotic effects of PFD on fibroblasts isolated from scarred VFs of ferrets.NA. Laryngoscope, 128:E171-E177, 2018.

To examine modulation of M-cadherin, a marker for satellite cells (SCs); and MyoD, which may indicate the myogenic activity following recurrent laryngeal nerve (RLN) denervation and immediate reinnervation; and to elucidate the correlation between their modulations and establishment of neuromuscular junctions (NMJs) in the reinnervated rat thyroarytenoid (TA) muscle.Quantitative real-time polymerase chain reaction qPCR and histologic assessment of the TA muscle following RLN transection and anastomosis.Rats were divided into three groups: 1) denervation alone (DNV) (n = 60), 2) denervation with anastomosis (ANS) (n = 60), and 3) sham-operated controls (n = 12). Animals were sacrificed at 3 days and 1, 3, and 5 weeks after treatment. TA muscles harvested from 40 animals from each DNV and ANS group; all of sham group were subjected to qPCR for assessment of the modulation of M-cadherin and MyoD; and the remaining larynges of DNV and ANS group were used for histologic analysis.The expression levels of messenger RNAs (mRNAs) encoding M-cadherin and MyoD in the TA muscle of the DNV group were significantly higher (P0.05) than in the control throughout the study period. These mRNA levels in the ANS group were significantly higher (P0.05) at ≤ 1 week than in the controls but fell to control levels at ≥ 3 weeks. In the ANS group, recovery of muscle area and NMJs structure occurred by 3 weeks.These data suggested that NMJ formation following reinnervation might prompt recovery of M-cadherin and MyoD mRNA expression to the quiescent level of SCs.

Objectives:Evaluate the long-term efficacy of laryngeal reinnervation with refined nerve-muscle pedicle (NMP) flap implantation combined with arytenoid adduction for unilateral vocal fold paralysis (UVFP) using laryngeal electromyography (LEMG), coronal images, and assessment of phonatory function.Methods:We retrospectively reviewed 16 UVFP patients who received refined NMP implantation with arytenoid adduction. Videostroboscopy, perceptual evaluation, acoustic analysis, aerodynamic analysis, LEMG, and coronal imaging were performed pre- and 2 years after surgery. For LEMG analysis, a four-point scale was employed to grade motor unit (MU) recruitment where 4+ represented absent recruitment, 3+ represented greatly decreased recruitment, 2+ represented moderately decreased recruitment, and 1+ represented mildly decreased activity with less than full interference pattern. Coronal images were assessed for differences in thickness of the vocal folds during phonation and inhalation.Results:Phonatory function re...

To evaluate the long-term efficacy of laryngeal reinnervation via refined nerve-muscle pedicle (NMP) flap implantation combined with arytenoid adduction to treat unilateral vocal fold paralysis (UVFP), employing laryngeal electromyography (LEMG), coronal imaging, and phonatory function assessment.Case series with chart review.University hospital.We retrospectively reviewed 12 UVFP patients who underwent refined NMP implantation with arytenoid adduction. Videostroboscopy, phonatory functional analysis, LEMG, and coronal imaging were performed before and 2 years after surgery. In LEMG analysis, a 4-point scale was employed to grade motor unit (MU) recruitment: 4+ reflected no recruitment, 3+ greatly decreased recruitment, 2+ moderately decreased recruitment, and 1+ mildly decreased activity, associated with less than the full interference pattern. Coronal images were assessed in terms of differences in thickness and the vertical positions of the vocal folds.Phonatory function improved significantly after operation in all patients. In terms of LEMG findings, the preoperative MU recruitment scores were 1+ in no patients, 2+ in 4 patients, 3+ in 1 patient, and 4+ in 7 patients. Postoperative MU recruitment results were 1+ in 6 patients, 2+ in 5 patients, 3+ in 1 patient, and 4+ in no patients. Thinning of the affected fold during phonation was evident preoperatively in 9 of 10 patients. The affected and healthy folds were equal in volume in 4 of 9 patients postoperatively.The LEMG findings and coronal imaging suggest that NMP implantation may have enabled successful reinnervation of the laryngeal muscles of UVFP patients.