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Introduction Revision framework surgeries might be required for unilateral vocal fold paralyses. However, outcomes and indications of revision surgeries have not been adequately documented. For a better understanding of indications for the procedure and to help in achieving better vocal outcomes, we performed a retrospective chart review of patients who underwent revision framework surgeries for unilateral vocal fold paralysis. Objectives This study aimed to present clinical features of patients who underwent revision framework surgeries for the treatment of unilateral vocal fold paralysis. Methods Of the 149 framework surgeries performed between October 2004 and October 2019, 21 revision framework surgeries were performed in 19 patients. Self-assessments by patients using the voice handicap index-10 questionnaire, and objective aerodynamic and acoustic assessments performed pre- and post-operatively were analyzed using the Wilcoxon’s signed-rank test for paired comparisons. Results Undercorrection was indicated as reasons for revision surgeries in all cases. The revision techniques included type I thyroplasty, type IV thyroplasty, and arytenoid adduction, and revision surgeries were completed without any severe complication in all cases. Pre- and post-operative voice handicap index-10 scores were obtained in 12 cases, and other parameters were evaluated in 18 cases. Significant improvements were observed in voice handicap index-10 scores, maximum phonation time, mean flow rate, Current/Direct Current ratio, and pitch perturbation quotient. Conclusion Undercorrection was observed in all patients who underwent revision framework surgeries for unilateral vocal fold paralysis, and the initial assessment and planning are thought to be important in order to avoid revision surgeries. Revision surgeries were performed safely in all cases, and significantly improved vocal outcomes were observed, even after multiple procedures. Revision surgery should be considered for patients with unsatisfactory vocal functions after primary framework surgeries for unilateral vocal fold paralysis.

Purpose: Auditory-perceptual evaluation is essential for the assessment of voice quality. The Consensus Auditory-Perceptual Evaluation of Voice (CAPE-V) provides a standardized protocol and assessment form for clinicians to analyze the voice quality and has been adapted into several different languages. The aims of this study were to develop the Japanese version of the CAPE-V and to investigate its reliability and validity. Method: The Japanese CAPE-V consisted of the same three speech contexts (vowels, sentences, and conversation) as developed in the original English version. The sentences were designed according to the concepts of the original version and reviewed by Japanese phoneticians. To validate the usefulness of the Japanese CAPE-V, voices of 173 Japanese-speaking subjects (76 subjects with dysphonia and 97 without voice complaints) were evaluated by five experienced judges, according to the Japanese CAPE-V as well as the GRBAS (Grade, Roughness, Breathiness, Asthenia, Strain) scale. Results: The Japanese CAPE-V provided a high interrater reliability (intraclass correlation coefficients [ICCs] > .85 for all the parameters) as well as a high intrarater reliability (ICCs > .85 for all the parameters). In addition, overall severity, roughness, and breathiness in the Japanese CAPE-V were highly correlated with the corresponding dimensions in the GRBAS scale, having Spearman correlation coefficients greater than .8. Conclusion: This study demonstrated the reliability and validity of the newly developed Japanese CAPE-V as an auditory-perceptual evaluation instrument.

Protein turnover is critical to cellular physiology as well as to the growth and maintenance of tissues. The unique synthesis and degradation rates of each protein help to define tissue phenotype, and knowledge of tissue- and protein-specific half-lives is directly relevant to protein-related drug development as well as the administration of medical therapies. Using stable isotope labeling and mass spectrometry, we determine the in vivo turnover rates of thousands of proteins—including those of the extracellular matrix—in a set of biologically important mouse tissues. We additionally develop a data visualization platform, named ApplE Turnover, that enables facile searching for any protein of interest in a tissue of interest and then displays its half-life, confidence interval, and supporting measurements. This extensive dataset and the corresponding visualization software provide a reference to guide future studies of mammalian protein turnover in response to physiologic perturbation, disease, or therapeutic intervention., Protein turnover underpins biology but is challenging to measure in vivo across the entire proteome. Here, the authors provide a comprehensive resource of protein turnover in mouse tissues and develop a visualization platform to analyze these data.

スターフルーツ（Averrhoa carambola L.）は沖縄や鹿児島などで栽培されている熱帯果樹である。本研究では，スターフルーツの着花数の増加と開花期の調節を目的として，環状剥皮，結縛，摘心および環状剥皮の幅の違いが着花に及ぼす影響について調査した。1年生の直立枝と下垂枝に分け，環状剥皮，結縛，摘心処理をそれぞれ行った結果，環状剥皮による着花促進がみられた。直立枝では花房数の有意な差はみられなかったが，下垂枝では花房数および小花数が最大であったことから，環状剥皮はスターフルーツにおいて着花促進効果が最も高い方法であると考えられた。さらに環状剥皮の幅の違いについて，1年生の枝に2.5mm, 5mm, 10mm幅でそれぞれ処理を行った。その結果，環状剥皮で着花促進効果がみられ，処理5週後の枝あたりの花房数が2.5mm幅区で最も多くなった。花房が着生している枝の割合は2.5mm, 5mm, 10mm幅区で90%以上に達した。これらの結果から，着花数増加のためにスターフルーツの枝に環状剥皮を行う場合，処理幅は2.5mmで十分であることが示された。, Star fruit (Averrhoa carambola L.) is a tropical fruit tree indigenous to Java and the Moluccas Islands that bears fruit throughout the year when cultivated under tropical conditions. In Japan, it is cultivated in Okinawa and the Kyushu region and has two annual flowering periods : one from May to June and the other from September to October. Cultivation of this species has been hindered by the lack of established cultivation methods, and because it produces few flowers. Consequently, controlling the flowering period and increasing the number of flowers would be advantageous for improving star fruit production. The influence of girdling, strapping, and pinching treatments to increase flower setting were investigated in different treatment plots in a vinyl greenhouse at Tokyo University of Agriculture, Kanagawa, Japan. The treatments were performed on upright and descending branches of starfruit. Descending branches had larger numbers of flower clusters than upright branches. Compared to the control, girdling significantly increased the number of flower clusters on descending branches, but no such effect was observed on upright branches. In addition, the influence of girdling width (2.5, 5, and 10 mm) on flower setting on horizontal branches was examined. Compared to the control, the number of flower clusters increased for all girdling widths. The largest numbers of flower clusters and small flowers were observed in the 2.5 mm width girdling plot at 5 weeks after treatment. For the 2.5, 5, and 10 mm width girdling plots, more than 90% of branches had flower clusters at 5 weeks after treatment, compared to 30% in the control plot. The findings demonstrate that girdling improves flower setting in star fruit.

ムラサキセンダイハギの花芽形成過程と低温遭遇時の開花反応について観察した。10月から8℃12週間の低温処理を行った後，最低温度15℃を維持した温室で栽培した。その結果，花芽形成過程は他のマメ科植物と類似しており，低温処理開始から4週間後には小花の分化が確認され，その時点でほぼ花芽となるシュート数は決定していた。次に8℃低温処理期間を0，8，12，16週間と変えて最低温度15℃を維持して栽培した区，および戸外に置いた自然低温区について生育・開花反応をみた。その結果，低温無処理（0週）では発蕾はしたものの開花には至らず，一方，8℃ 12週間の低温処理は自然低温区と比べて開花が50日以上早まった。, We observed the process of flower bud formation, and investigated the flowering response of blue wild indigo exposed to low temperature. After low-temperature treatment at 8℃ for 12 weeks from October, the plants were cultivated in a greenhouse maintained at a minimum temperature of 15℃. The process of flower bud initiation and development was similar to the plant of Fabaceae and the florets was differentiated until 4 weeks after low temperature treatment. The number of floral shoots was almost all determined at that time. The growth and flowering response were observed in cultivation at a minimum temperature of 15℃ treated after 0, 8, 12, and 16 weeks at 8℃, and the natural low temperature plot. Flowering was accelerated 50 days and more by low-temperature treatment at 8℃ for 12 weeks compared to the natural low temperature plot.

To evaluate whether readout-segmented echo-planar imaging (RS-EPI) diffusion-weighted imaging (DWI) can reduce image distortion and improve the lesion identification in parathyroid adenomas (PTAs) compared to single-shot EPI (SS-EPI) DWI, and to determine whether PTAs can be differentiated from other soft tissue structures of the head and neck region by using the apparent diffusion coefficient (ADC) value.We retrospectively analyzed the preoperative MR images including DWI of 24 patients with surgically confirmed PTA. RS-EPI and SS-EPI DWI were evaluated by two independent readers for the identification of the lesions and distortion. The ADC values of the PTAs were compared with those of thyroid glands and cervical lymph nodes.RS-EPI provided significantly less distortion compared to SS-EPI. RS-EPI tended to have better lesion identification compared with SS-EPI without a statistically significant difference. On SS-EPI, the PTAs had significantly higher ADC values compared with the cervical lymph nodes. On RS-EPI, the PTAs had significantly higher ADC values compared with the thyroid glands and cervical lymph nodes.RS-EPI reduces the DWI distortion in PTAs. The ADC value obtained using RS-EPI enables the differentiation of PTAs from nearby structures, such as thyroid glands and cervical lymph nodes.

Post-radiation fibrosis of the vocal folds is thought to cause vocal impairment. However, the mechanism by which this occurs has been poorly documented, probably because of the lack of an appropriate experimental animal model. The purpose of this study was to establish a simple and reproducible mouse model of laryngeal radiation to investigate the development of vocal fold fibrosis over time. C57BL/6 mice individually placed in a lead shield were irradiated with a single dose of 20 Gy. At 1, 2, and 6 months after irradiation, larynges were harvested and subjected to histological examination and gene expression analysis. Irradiated vocal folds showed time-dependent tissue contraction and increased collagen deposition, with no significant difference in the changes in hyaluronic acid levels. Transcriptional analysis revealed upregulated expressions of TGF-β1 and iNOS at 6 months, but downregulated expressions of Acta2, Col1a1, Col3a1, and MMP8. Moreover, elevated TGF-β1 and reduced downstream gene expression levels indicated the existence of an inhibitory factor over the TGF-β/Smad pathway. Discrepancies in histological and transcriptional studies of collagen might suggest that radiation-induced vocal fold fibrosis could be caused by the elongated turnover of collagen. Overall, we established a mouse model of radiation-induced vocal fold fibrosis using a simple protocol. Further investigations are warranted to elucidate the pathogenesis of irradiation-induced fibrosis in vocal folds.

Macrophages aid in wound healing by changing their phenotype and can be a key driver of fibrosis. However, the contribution of macrophage phenotype to fibrosis following vocal fold injury remains unclear. Peroxisome proliferator-activated receptor-γ (PPARγ) is expressed mainly by macrophages during early wound healing and regulates the macrophage phenotype. This study aimed to evaluate the effects of pioglitazone, a PPARγ agonist, on the macrophage phenotype and fibrosis following vocal fold injury in rats. Pioglitazone was injected into the rats' vocal folds on days 1, 3, 5, and 7 after injury, and the vocal fold lamina propria was evaluated on days 4 and 56 after injury. Moreover, THP-1-derived macrophages were treated with pioglitazone, and the expression of pro-inflammatory cytokines under lipopolysaccharide/interferon-γ stimulation was analyzed. The results revealed that pioglitazone reduced the expression of Ccl2 both in vivo and in vitro. Furthermore, pioglitazone decreased the density of inducible nitric oxide synthase+ CD68+ macrophages and inhibited the expression of fibrosis-related factors on day 4 after injury. On day 56 after injury, pioglitazone inhibited fibrosis, tissue contracture, and hyaluronic acid loss in a PPARγ-dependent manner. These results indicate that PPARγ activation could inhibit accumulation of inflammatory macrophages and improve tissue repair. Considered together, these findings imply that inflammatory macrophages play a key role in vocal fold fibrosis.

Objective We investigated the efficacy of non-contrast 3-Tesla MR imaging added to the combination of sestamibi with99mTc (MIBI) scintigraphy and Ultrasonography (US) for the pre-operative localization of Primary Hyperparathyroidism (PHPT) lesions. Methods A total of 34 parathyroid glands, including nine normal glands, were examined with MIBI, US, and non-contrast 3-Tesla MRI. MRI was performed with the acquisition of T1- and T2-weighted images and fat-suppressed T2-weighted images. We calculated the sensitivities of MIBI, US, and the 'additional' MRI, with knowledge of the former two modalities' results. Results For the diagnosis of PHPT lesions, the sensitivity values of MIBI, US, and additional MRI were 88.0% (22/25), 84.0% (21/25), and 92.0% (23/25), respectively. Normal glands were not visualized with any modality (0/9). One lesion was detected neither with US nor MRI, but only with MIBI, with the limitation that MIBI represented no more than laterality. The two glands not identified in MRI were 4 mm and 6 mm in their size, which are within the range of normal gland's size. Two lesions were not detected with US or MIBI but were visualized with the additional MRI, which indicated that the MRI contributed an 8.0% (2/25) improvement of sensitivity, compared from that of US. Fat-suppressed T2-weighted images were useful in the identification of parathyroid lesions, as these images helped to differentiate between the lesion and the adjacent tissue. Conclusion Additional non-contrast 3-Tesla MRI was a useful adjunctive tool for localization of PHPT, which improved the sensitivity of the pre-operative localization of PHPT lesions. Fat-suppressed T2-weighted images contributed to their identification. Level vi Evidence from a single descriptive or qualitative study.

The laryngotracheal cartilage is a cardinal framework for the maintenance of the airway for breathing, which occasionally requires reconstruction. Because hyaline cartilage has a poor intrinsic regenerative ability, various regenerative approaches have been attempted to regenerate laryngotracheal cartilage. The use of autologous mesenchymal stem cells (MSCs) for cartilage regeneration has been widely investigated. However, long-term culture may limit proliferative capacity. Human-induced pluripotent stem cell-derived MSCs (iMSCs) can circumvent this problem due to their unlimited proliferative capacity. This study aimed to investigate the efficacy of iMSCs in the regeneration of thyroid cartilage in immunodeficient rats. Herein, we induced iMSCs through neural crest cell intermediates. For the relevance to prospective future clinical application, induction was conducted under xeno-free/serum-free conditions. Then, clumps fabricated from an iMSC/extracellular matrix complex (C-iMSC) were transplanted into thyroid cartilage defects in immunodeficient rats. Histological examinations revealed cartilage-like regenerated tissue and human nuclear antigen (HNA)-positive surviving transplanted cells in the regenerated lesion. HNA-positive cells co-expressed SOX9, and type II collagen was identified around HNA-positive cells. These results indicated that the transplanted C-iMSCs promoted thyroid cartilage regeneration and some of the iMSCs differentiated into chondrogenic lineage cells. Induced MSCs may be a promising candidate cell therapy for human laryngotracheal reconstruction.

Objectives/hypothesis To elucidate the aging physiology of the vocal folds, we examined the characters of aged vocal fold fibroblasts (VFFs) in various conditions. Study design In vitro study. Methods VFFs from young (12-week-old) and aged (19-month-old) Sprague-Dawley rats were compared. Proliferative capacity, ratio of myofibroblast to fibroblast, myofibroblast function, and extracellular matrix production were examined in the following conditions: naive, basic fibroblast growth factor (bFGF) supplemented, and hepatocyte growth factor (HGF) supplemented. Results Aged VFFs demonstrated reduced proliferation by cell counting, though the ratio of Ki-67-positive cells showed no difference. Aged VFFs exhibited an increased expression of α-smooth muscle actin (α-SMA); however, they demonstrated no enhanced contractile ability in a gel contraction assay. Type I collagen protein was increased age dependently, accompanied with decreased Mmp1 and unchanged Col1a1 transcription. Type I collagen protein and α-SMA represented quite similar reduction patterns to bFGF or HGF administration. Conclusions The following possible characteristics of aged VFFs were implied: long duration of mitosis, increased myofibroblast population size with certain dysfunctions, reduced type I collagen turnover, and correlation between α-SMA expression and type I collagen metabolism. Further investigations of these features will help to clarify presbyphonia's pathology and establish treatment strategies. Level of evidence NA Laryngoscope, 129:E94-E101, 2019.

Objectives/Hypothesis This study aimed to reveal the effects of adipose-derived mesenchymal stromal cells (ASCs) on prevention of vocal fold scarring by investigating how the immediate ASCs transplantation into the injured rat vocal fold affect the levels of gene transcription and translation. Study Design Prospective animal experiments with controls. Methods ASCs harvested from green fluorescent protein transgenic rat (ASCs group) or saline (sham group) were injected into the thyroarytenoid muscle of Sprague-Dawley rats immediately after stripping the vocal fold. For histological examinations, larynges were extirpated at 3, 14, and 56 days after the injection. Quantitative real-time polymerase chain reaction (PCR) analyses were performed at 3 and 14 days after the injection. Results Transplanted ASCs were detected only in larynges at day 3. At days 14 and 56, histological examination showed significantly higher amounts of hyaluronic acid and lower deposition of collagen in the ASCs group compared to the sham group. Real-time PCR revealed that the ASCs group showed low expression of procollagen (Col)1a1, Col1a3, matrix metalloproteinase (Mmp)1 and Mmp8 in each time points. The ASCs group showed high expression of fibroblast growth factor (Fgf)2 and Hepatocyte growth factor (Hgf) compared to the sham group at day 14. Conclusions ASCs increased expressions of Fgf2 and Hgf, and suppressed excessive collagen deposition during vocal fold wound healing. Given the fact that ASCs survived no more than 14 days, ASCs were thought to induce upregulations of growth factors' genes in surrounding cells. These results suggested that ASCs have potential to prevent vocal fold scarring. Level of Evidence NA. Laryngoscope, 128:E33–E40, 2018

Objectives/Hypothesis Vocal fold scarring, which causes severe hoarseness, is intractable. The optimal treatment for vocal fold scarring has not been established; therefore, prevention of scarring is important. The aim of this study was to clarify the effectiveness of basic fibroblast growth factor (bFGF) for prevention of postsurgical vocal fold scarring. Study Design Prospective animal experiments with controls. Method The vocal folds of Sprague-Dawley rats were injured unilaterally or bilaterally after local application of a 10 μL solution of bFGF. Larynges ware harvested for histological and immunohistochemical examination 2 months postoperation and for quantitative real-time polymerase chain reaction (qRT-PCR) analysis 1 week postoperation. Results Histological examination showed significantly increased hyaluronic acid and decreased deposition of dense collagen in the bFGF-treated group at 100 ng/10 μL compared with the sham-treated group. Immunohistochemical examination showed significantly decreased collagen type III deposition in the bFGF-treated group at 100 ng/10 μL compared with the sham-treated group. qRT-PCR revealed that hyaluronan synthase 2 (Has2), Has3, and hepatocyte growth factor were upregulated in bFGF-treated groups compared with sham-treated group. Conclusion The current results suggest that local application of bFGF at the time of injury has the potential to prevent vocal fold scarring. Preventive injection of bFGF could be applied at the time of phonomicrosurgery to avoid postoperative scar formation. Level of Evidence N/A. Laryngoscope, 2016 127:E67–E74, 2017

Objectives/Hypothesis Stem cells are known to proliferate at a slow rate in adult organs, and thus slow-cycling cells exhibiting pluripotency are considered tissue-specific stem cells in some organs. Slow-cycling cells in the vocal fold (VF) have not been well documented. Here we sought to clarify the distributions and characteristics of slow-cycling cells in rat VFs. Methods We applied double-labeling technique to detect the distribution of slow-cycling cells. We injected the exogenous proliferation marker 5-bromo-2'-deoxyuridine (BrdU) into Sprague-Dawley rats. After a chasing period, VFs were immunostained with antibodies to BrdU and the second endogenous proliferation marker, Ki-67. BrdU (+) Ki-67(+) cells were regarded as slow-cycling cells and counted by VF regions. To reveal slow-cycling cells' characteristic, their immunophenotypes were histologically investigated and their kinetics in injured VFs were evaluated. Results Most slow-cycling cells were detected in the basal layer of the epithelium. Slow-cycling cells in the epithelium displayed a low positive ratio of E-cadherin and CK5 and a high positive ratio of vimentin and CD31 as compared with the other epithelial cells. The expression of S100A4 was low in slow-cycling cells of the lamina propria and the macula flava. FGFR1, HAS1, HAS2, and HAS3 were not detected in the slow-cycling cells. A time-dependent reduction of slow-cycling cells was observed in injured VFs. Conclusion Most slow-cycling cells resided in the epithelium, exhibiting various phenotypes in a relatively undifferentiated condition, and they are suspected to contribute to the tissue repair of the injured VFs. Level of Evidence N/A. Laryngoscope, 126:E164–E170, 2016

In the management of laryngeal pre-cancerous lesions such as dysplasia or carcinoma in situ (CIS), it is important that lesion regression occur without any complications. As a minimally invasive treatment option, photocoagulation therapy using angiolytic lasers has been attracting attention. Therapeutic effects have been reported for this type of treatment, however, vocal function after treatment has not been well discussed. In this retrospective case series, we examined the therapeutic effects of photocoagulation therapy on laryngeal dysplasia and the impact on vocal function. Twenty-four patients with laryngeal dysplasia or CIS were treated with photocoagulation therapy using angiolytic lasers. Two patients were treated under general anesthesia, the remaining 22 patients were treated with topical anesthesia. Before and after treatment the extent of the lesion and vocal function was evaluated by endoscopic examination and acoustic and aerodynamic analyses, respectively. More than 50 % disease regression was observed in 20 of 24 patients. Acoustic and aerodynamic analyses revealed improvement in pitch perturbation quotient with no impairment in other parameters. Photocoagulation therapy using angiolytic lasers has proven to be feasible and safe for the treatment of laryngeal dysplasia.

To elucidate the aging physiology of the vocal folds, we examined the characters of aged vocal fold fibroblasts (VFFs) in various conditions.In vitro study.VFFs from young (12-week-old) and aged (19-month-old) Sprague-Dawley rats were compared. Proliferative capacity, ratio of myofibroblast to fibroblast, myofibroblast function, and extracellular matrix production were examined in the following conditions: naïve, basic fibroblast growth factor (bFGF) supplemented, and hepatocyte growth factor (HGF) supplemented.Aged VFFs demonstrated reduced proliferation by cell counting, though the ratio of Ki-67-positive cells showed no difference. Aged VFFs exhibited an increased expression of α-smooth muscle actin (α-SMA); however, they demonstrated no enhanced contractile ability in a gel contraction assay. Type I collagen protein was increased age dependently, accompanied with decreased Mmp1 and unchanged Col1a1 transcription. Type I collagen protein and α-SMA represented quite similar reduction patterns to bFGF or HGF administration.The following possible characteristics of aged VFFs were implied: long duration of mitosis, increased myofibroblast population size with certain dysfunctions, reduced type I collagen turnover, and correlation between α-SMA expression and type I collagen metabolism. Further investigations of these features will help to clarify presbyphonia's pathology and establish treatment strategies.NA Laryngoscope, 129:E94-E101, 2019.

Objectives/Hypothesis Vocal fold scar remains a therapeutic challenge. Mesenchymal stromal cells (MSCs) are promising tools for regenerative medicine. Nevertheless, few in vivo studies have directly compared various sources of MSCs. The aim of this study was to investigate the therapeutic potential of adipose-derived stromal cells (ASCs) in comparison with bone marrow–derived stromal cells (BMSCs) for vocal fold regeneration. Study Design Prospective animal experiments with controls. Methods Two months after stripping of the lamina propria, 18 beagles were divided into four implantation groups: atelocollagen alone (collagen group), atelocollagen with BMSCs (BMSC-collagen), atelocollagen with ASCs (ASC-collagen), or a sham-treated group. One or 6 months after implantation, vibratory and histological examinations were performed. Results Mucosal vibration was significantly improved in both of the MSC-implanted groups compared with the sham-treated group, whereas only the ASC-collagen group showed a significantly smaller glottal gap than the collagen group. Moreover, in the ASC-collagen group, a significant reduction of collagen density was observed compared to the sham-treated group, and there was a trend for better restoration of hyaluronic acid (HA). Implanted MSCs were detected 1 month postimplantation; however, none survived 6 months postimplantation. Conclusions Although implantation of an atelocollagen sponge and ASCs into vocal fold scars induced vibratory recovery comparable to that of BMSCs, ASCs might have more potential in terms of restoration of HA and suppression of excessive collagen deposition. Level of Evidence NA Laryngoscope, 2015

Vocal fold scar remains a therapeutic challenge. Basic fibroblast growth factor (bFGF) was reported to have regenerative effects for vocal fold scar, although it has the disadvantage of rapid absorption in vivo. A collagen-gelatin sponge (CGS) can compensate for the disadvantage by providing a sustained release system. The current study evaluated the efficacy of CGS combined with bFGF on vocal fold scar, using rat fibroblasts for an in vitro model and a canine in vivo model. We prepared fibroblasts from scarred vocal folds (sVFs) in rats and showed that bFGF accelerated cell proliferation and suppressed expression levels of cleaved caspase 3 and α-smooth muscle actin. Has 1, Has 3, Fgf2, Hgf and Vegfa mRNA levels were significantly upregulated, while Col1a1 and Col3a1 were dose-dependently downregulated, with a maximum effect at 100 ng/ml bFGF. In an in vivo assay, 6 weeks after lamina propria stripping, beagles were divided into three groups: CGS alone (CGS group); CGS with bFGF (7 µg/cm2 ; CGS + bFGF group); or a sham-treated group. Vibratory examination revealed that the glottal gap was significantly reduced in the bFGF group and the two implanted groups, whereas the CGS + bFGF group showed higher mucosal wave amplitude. Histological examination revealed significantly restored hyaluronic acid and elastin redistribution in the CGS + bFGF group and reductions in dense collagen deposition. These results provide evidence that CGS and bFGF combination therapy may have therapeutic potential and could be a promising tool for treating vocal fold scar. Copyright © 2015 John Wiley & Sons, Ltd.

Vocal fold scar and sulcus are intractable diseases with no effective established treatments. Hepatocyte growth factor (HGF) has preclinically proven to have potent antifibrotic and regenerative effects on vocal fold scar. The current Phase I/II clinical trial aims to examine the safety and effectiveness of intracordal injection of a recombinant human HGF drug for patients with vocal fold scar or sulcus. This is an open-label, dose-escalating, first-in-human clinical trial. Eighteen patients with bilateral vocal fold scar or sulcus were enrolled and divided into three groups: Step I received 1 μg of HGF per vocal fold; Step II received 3 μg of HGF; and Step III received 10 μg of HGF. Injections were administered once weekly for 4 weeks. The protocol treatment was performed starting with Step I and escalating to Step III. Patients were followed for 6 months post-treatment. Local and systemic safety aspects were examined as primary endpoints, and therapeutic effects were assessed as secondary endpoints using voice handicap index-10; maximum phonation time; vocal fold vibratory amplitude; grade, rough, breathy, asthenic, strained scale; and jitter. The results indicated no serious drug-related adverse events in either the systemic or local examinations. In whole-subject analysis, voice handicap index-10, vocal fold vibratory amplitude, and grade, rough, breathy, asthenic, strained scale were significantly improved at 6 months, whereas maximum phonation time and jitter varied. There were no significant differences in phonatory data between the step groups. In conclusion, intracordal injection of a recombinant human HGF drug was safe, feasible, and potentially effective for human patients with vocal fold scar or sulcus.

Summary Objectives Professional voice users, such as singers and teachers, are at greater risk of developing vocal fold injury from excessive use of voice; thus, protection of the vocal fold is essential. One of the most important factors that aggravates injury is the production of reactive oxygen species at the wound site. The purpose of the current study was to assess the effect of astaxanthin, a strong antioxidant, on the protection of the vocal fold from injury and inflammation due to vocal loading. Study Design This study is an institutional review board-approved human clinical trial. Methods Ten male subjects underwent a 60-minute vocal loading session and received vocal assessments prior to, immediately after, and 30 minutes postvocal loading (AST(−) status). All subjects were then prescribed 24 mg/day of astaxanthin for 28 days, after which they received the same vocal task and assessments (AST(+) status). Phonatory parameters were compared between both groups. Results Aerodynamic assessment, acoustic analysis, and GRBAS scale (grade, roughness, breathiness, asthenia, and strain) were significantly worse in the AST(−) status immediately after vocal loading, but improved by 30 minutes after loading. In contrast, none of the phonatory parameters in the AST(+) status were statistically worse, even when measured immediately after vocal loading. No allergic responses or adverse effects were observed after administration of astaxanthin. Conclusions The current results suggest that astaxanthin can protect the vocal fold from injury and inflammation caused by vocal loading possibly through the regulation of oxidative stress.

Vocal fold scarring, which causes severe hoarseness, is intractable. The optimal treatment for vocal fold scarring has not been established; therefore, prevention of scarring is important. The aim of this study was to clarify the effectiveness of basic fibroblast growth factor (bFGF) for prevention of postsurgical vocal fold scarring.Prospective animal experiments with controls.The vocal folds of Sprague-Dawley rats were injured unilaterally or bilaterally after local application of a 10 μL solution of bFGF. Larynges ware harvested for histological and immunohistochemical examination 2 months postoperation and for quantitative real-time polymerase chain reaction (qRT-PCR) analysis 1 week postoperation.Histological examination showed significantly increased hyaluronic acid and decreased deposition of dense collagen in the bFGF-treated group at 100 ng/10 μL compared with the sham-treated group. Immunohistochemical examination showed significantly decreased collagen type III deposition in the bFGF-treated group at 100 ng/10 μL compared with the sham-treated group. qRT-PCR revealed that hyaluronan synthase 2 (Has2), Has3, and hepatocyte growth factor were upregulated in bFGF-treated groups compared with sham-treated group.The current results suggest that local application of bFGF at the time of injury has the potential to prevent vocal fold scarring. Preventive injection of bFGF could be applied at the time of phonomicrosurgery to avoid postoperative scar formation.N/A. Laryngoscope, 2016 127:E67-E74, 2017.

カキ‘富有’と‘次郎’を用いて，側枝への環状剥皮と結縛の処理およびそれらとサイトカイニン活性をもつ合成化合物のCPPUの果実塗布の組み合わせ処理が果実品質に及ぼす影響について調べた．‘富有’では環状剥皮の5月と7月処理で，結縛の5月，6月および7月処理で果実重が増加した．果実の糖度は，5月，6月および7月の結縛処理により増加した．これらの効果は環状剥皮処理に比べ結縛処理の方が大きかった．‘次郎’では6月の環状剥皮処理で果実重と果径が無処理に比べ大きかった．処理時期としては5月または6月処理の果実品質が良好であったことから，果実生育ステージの第I期前半が適期と推察された．‘富有’では，環状剥皮とCPPUの組み合わせで最も果実肥大が大きかった．‘次郎’では，CPPUは単独，組み合わせ処理ともに果実の肥大を抑制した．また，‘富有’，‘次郎’ともに，CPPU処理により糖度は低下し，着色抑制がみられた．

Vocal fold scar remains a therapeutic challenge. Mesenchymal stromal cells (MSCs) are promising tools for regenerative medicine. Nevertheless, few in vivo studies have directly compared various sources of MSCs. The aim of this study was to investigate the therapeutic potential of adipose-derived stromal cells (ASCs) in comparison with bone marrow-derived stromal cells (BMSCs) for vocal fold regeneration.Prospective animal experiments with controls.Two months after stripping of the lamina propria, 18 beagles were divided into four implantation groups: atelocollagen alone (collagen group), atelocollagen with BMSCs (BMSC-collagen), atelocollagen with ASCs (ASC-collagen), or a sham-treated group. One or 6 months after implantation, vibratory and histological examinations were performed.Mucosal vibration was significantly improved in both of the MSC-implanted groups compared with the sham-treated group, whereas only the ASC-collagen group showed a significantly smaller glottal gap than the collagen group. Moreover, in the ASC-collagen group, a significant reduction of collagen density was observed compared to the sham-treated group, and there was a trend for better restoration of hyaluronic acid (HA). Implanted MSCs were detected 1 month postimplantation; however, none survived 6 months postimplantation.Although implantation of an atelocollagen sponge and ASCs into vocal fold scars induced vibratory recovery comparable to that of BMSCs, ASCs might have more potential in terms of restoration of HA and suppression of excessive collagen deposition.NA Laryngoscope, 126:1143-1150, 2016.

Stem cells are known to proliferate at a slow rate in adult organs, and thus slow-cycling cells exhibiting pluripotency are considered tissue-specific stem cells in some organs. Slow-cycling cells in the vocal fold (VF) have not been well documented. Here we sought to clarify the distributions and characteristics of slow-cycling cells in rat VFs.We applied double-labeling technique to detect the distribution of slow-cycling cells. We injected the exogenous proliferation marker 5-bromo-2'-deoxyuridine (BrdU) into Sprague-Dawley rats. After a chasing period, VFs were immunostained with antibodies to BrdU and the second endogenous proliferation marker, Ki-67. BrdU (+) Ki-67(+) cells were regarded as slow-cycling cells and counted by VF regions. To reveal slow-cycling cells' characteristic, their immunophenotypes were histologically investigated and their kinetics in injured VFs were evaluated.Most slow-cycling cells were detected in the basal layer of the epithelium. Slow-cycling cells in the epithelium displayed a low positive ratio of E-cadherin and CK5 and a high positive ratio of vimentin and CD31 as compared with the other epithelial cells. The expression of S100A4 was low in slow-cycling cells of the lamina propria and the macula flava. FGFR1, HAS1, HAS2, and HAS3 were not detected in the slow-cycling cells. A time-dependent reduction of slow-cycling cells was observed in injured VFs.Most slow-cycling cells resided in the epithelium, exhibiting various phenotypes in a relatively undifferentiated condition, and they are suspected to contribute to the tissue repair of the injured VFs.N/A.

The mechanism of flower thinning by calcium formate was investigated in the Japanese pear cv. Kosui (Pyrus pyrifolia Nakai). Calcium formate thinned 30-40% flowers when a 1% solution was applied to the pistils just before or 24 hours after pollination, whereas 1% calcium acetate and calcium lactate were ineffective. Both adhesion of pollen grains to stigmatic surface and tube elongation in the style were reduced by calcium formate application. Calcium formate at 0.05% or higher concentrations was a much stronger inhibitor on pollen germination in vitro compared to calcium acetate or calcium lactate. When the inhibitory effect of formic acid, acetic acid, and lactic acid in vitro on pollen germination was compared, formic acid was the strongest inhibitor, suggesting that the compound induces floral abscission by preventing fertilization through the action of formate. At harvest, there was little or no difference in size and sugar content between the treated and control fruits. Thus, calcium formate may be a promising chemical thinner for Japanese pear flowers by reducing the cost of hand thinning.

海洋深層水の園芸利用を目的としてウンシュウミカン並びにトマトに施用し，果実への影響について検討した．本実験では，各々の栽培土壌に直接散布した．その結果，ウンシュウミカンでは，果実のサイズは小さくなり，糖度の増加が認められた．トマトでは，果実のサイズは小さくなり，糖と酸の両方が高くなり，果実の内部が緑色を呈した．これらの結果は塩類が大きく作用していると考えられるが，トマト果実内部の色の違いの原因はわからない．

Vocal fold scar remains a therapeutic challenge. Basic fibroblast growth factor (bFGF) was reported to have regenerative effects for vocal fold scar, although it has the disadvantage of rapid absorption in vivo. A collagen-gelatin sponge (CGS) can compensate for the disadvantage by providing a sustained release system. The current study evaluated the efficacy of CGS combined with bFGF on vocal fold scar, using rat fibroblasts for an in vitro model and a canine in vivo model. We prepared fibroblasts from scarred vocal folds (sVFs) in rats and showed that bFGF accelerated cell proliferation and suppressed expression levels of cleaved caspase 3 and α-smooth muscle actin. Has 1, Has 3, Fgf2, Hgf and Vegfa mRNA levels were significantly upregulated, while Col1a1 and Col3a1 were dose-dependently downregulated, with a maximum effect at 100 ng/ml bFGF. In an in vivo assay, 6 weeks after lamina propria stripping, beagles were divided into three groups: CGS alone (CGS group); CGS with bFGF (7 µg/cm

Anthocyanin synthesis began in the skin of ‘Olympia’ ( Vitis labruscana Bailey) grape when the berry growth reached a late stage in the second sigmoid growth-curve. During berry development, shikimate NADP oxidoreductase (SORase, EC.1.1.1.25), phenylalanine ammonia-lyase (PAL, EC.4.3.1.5), and chalcone flavanone isomerase (CHFI, EC.5.5.1.6) were investigated in the skin. These enzyme activities were high during early development of the young berry, but rapidly decreased thereafter, and were activated again when rapid anthocyanin synthesis subsequently occurred. When PAL from the young berry was analyzed by isoelectricfocusing polyacrylamide gel electrophoresis (IEF-PAGE), it showed a single activity band with a p I value of approximately 5.4, whereas PAL from the ripe berry showed two activity bands with p I s of 5.4 and 6.5. Thus, SORase, PAL, and CHFI seem to be involved in anthocyanin accumulation simultaneously, and newly synthesized isozyme of PAL may play a key role in pigment formation in ‘Olympia’ skin. The function of these enzyme activities in young berry is also discussed in this text.

Anthocyanin biosynthesis occurred in the half-cut grape berry ( Vitis labruscana Bailey, cv. Olympia) cultured in vitro under a continuous irradiance of 68 μmol m −2 s −1 white light. The use of 1 g l −1 abscisic acid (ABA) as a culture solution induced 2.5 times higher pigmentation than control (H 2 O) after 5-day culture. Rhamnose (10%) also accelerated pigmentation significantly, but 10% sucrose inhibited it. When ABA was mixed with rhamnose, the promotive effect was pronounced synergistically. In the skin of berry cultured with ABA, soluble sugar content and activity of chalcone flavanone isomerase (CHFI, E.C. 5.5.1.6) increased. Thus, ABA, some kinds of sugars, and anthocyanin biosynthetic enzymes in the skin are predominant determinants, respectively, for berry coloration.

The S-allele-associated proteins (S-proteins) in the styles of the Japanese pear (Pyrus serotina Rehd. var. culta Rehd.) were purified by cation exchange chromatography. Their inhibitory action on the growth of incompatible pollen tubes (pollen tubes bearing the same S- allele as in the style from which the S-proteins were prepared) was characterized in vitro. Germination and tube growth of self-pollen (pollen from the same cultivar from which the S-proteins were prepared) decreased dose-dependently when the S-protein was added to the medium. Tube length was reduced to 10% that of compatible pollen tubes (pollen tubes bearing the S-allele different from that in the style from which the S-proteins were prepared) at 1.5 µg µl1. S-proteins from Shinsui (S 4 S 5 ) also inhibited growth of cross-incompatible Kosui (S 4 S 5 ) pollen tubes, but not of compatible Chojuro (S 2 S 3 ) pollen tubes. After inactivation of RNase of the S- protein, the inhibitory action of the S-protein disappeared. These results indicate that the S-protein acts directly to inhibit growth of incompatible pollen tubes in Japanese pear styles, and that the RNase activity of the protein is essential for the biological function. However, small amounts of proteins that co-migrated with the S-protein may also play some roles in the inhibition. This is the first report on the selective inhibitory action of S-proteins in Rosaceae.

Self-incompatibility genotypes (S-genotype) were reexamined in the Japanese pear 'Choju', 'Kimizukawase', 'Meigetsu', and 'Ichiharawase'. The S-genotypes of the former 2 cultivars are designated as S2S5, whereas those of the latter 2 cultivars are S1S5. 'Choju' and 'Kimizukawase', which are cross-incompatible in both directions, possessed the S1- and S5-allele associated proteins (S-protein) in their styles. PCR (polymerase chain reaction) amplifications of self-incompatibility genes (S-gene) also revealed the presence of S1- and S5-gene fragments in both cultivars. Thus, the S-genotypes of 'Choju' and 'Kimizukawase' were confirmed to be S1S5, but not S2S5. 'Meigetsu' and 'Ichiharawase' were compatible with 'Choju' ; their styles contained the S1-protein but not the S5-protein. PCR amplification also suggested the absence of S5-gene in both cultivars. These results together with those in the literature on the appearance of F1 individuals with pollen cross-incompatibility with 'Meigetsu' indicate that the S-genotypes of 'Meigetsu' and 'Ichiharawase' are S1S7 or S1Sx (Sx indicates an allele other than S1 to S7).

An S4-allele-associated protein (S4-protein) was identified by both isoelectricfocusing (IEF) polyacrylamide-gel electrophoresis and N-terminal amino-acid sequences in self-incompatible Nijisseiki (self-in- compatibility genotype=S2S4) and self-compatible Osa-Nijisseiki (S2S4 SM, SM=stylar-part mutant) styles of the Japanese pear, Pyrus serotina Rehd. var. culta Rehd. Expression of the protein in the cultivars was compared during flower bud development and at post-transcriptional levels. In mature styles, S4-protein could be detected on IEF gel in both cultivars and the N-terminal amino acid sequences were identical, although Osa-Nijisseiki contained only one-third the amount contained in Nijisseiki. No difference was observed in S2-protien amounts. In Nijisseiki styles, S4-protein was already detectable 8 days before anthesis (DBA) and it was synthesized consistently until 2 days after anthesis (DAA); the amount increased 4.7-fold during these 10 days. In contrast, S4-protein in Osa-Nijisseiki was not detected earlier than 6 DBA; a small amount was found at 4 DBA, and it increased gradually as flowers developed. Thus, expression of Osa-Nijisseiki S4-protein is developmentally controlled in the same way as that of Nijisseiki S4-protein, but with a time lag of several days; the protein level at 2 DAA corresponded to that of Nijisseiki earlier than 4 DBA. S4-proteins from both Nijisseiki and Osa- Nijisseiki showed RNase activity and the activity was also developmentally controlled; it increased about fourfold during the interval from 8 DBA to 2 DAA in Nijisseiki, and 3.3-fold during the interval from 4 DBA to 2 DAA in Osa-Nijisseiki. Activity at 2 DAA, however, was twice as high in Nijisseiki. In vitro protein synthesis showed that poly(A)+ from mature Osa-Nijisseiki styles could form S4-like protein in a manner similar to that of Nijisseiki. These results suggest that the self-compatibility of Osa-Nijisseiki is due to a low level of S4-protein expression, a mechanism very similar to the low level of S-protein and weak incompatibility in immature styles of self-incompatible Nijisseiki. Part of this protein repression may be regulated during post-transcriptional events.

The effects of applications of 6-benzylaminopurine (BAP), gibberellic acid (GA3), and abscisic acid (ABA) on the root formation of grapevine hardwood cuttings were investigated. BAP suppressed rooting of grapevine cuttings completely, whereas GA3 and ABA inhibited it temporarily. Endogenous indoleacetic acid (IAA) in cuttings treated with BAP increased linearly to 12.6 and 19.4 times per cutting and per g fresh weight, respectively, over the control for 30 days after planting. In cuttings treated with GA3, the level of diffusible IAA was higher than that of control cuttings through the experimental period; the fluctuation pattern of extractable IAA was similar to that of the control. In cuttings treated with ABA, the extractable IAA content was significantly lower than that in control cuttings 10 days after planting. The fluctuation pattern of endogenous IAA in BAP treatment was different from that in GA3 and ABA treatments. Cuttings treated with BAP presumably failed to root because the level of endogenous IAA was unbalanced. GA3 may inhibit root formation directly whereas ABA may cause inhibition of rooting by depressing the bud activity.

The effect of different severities of root flooding at budbreak and 70 days after budbreak (8 to 12 leaf stage) on root and shoot growth of 'AXR#1', 'St. George', '11OR', and '420A' rootstocks was investigated. The four severities of root flooding were control (0%), 33%, 67%, and 100% of the root soil volume of each rootstock. At the budbreak stage, root and shoot growths of all rootstocks were strongly inhibited when two-thirds or more of the root soil volume was flooded. At 100% root flooding, there was no new root growth. The number of buds that sprouted and developed was unaffected by flooding. Within 10 to 15 days after flooding, the cuttings at 8 to 12 leaf stage of '420A' and '11OR' grew poorly. The color of roots exposed to 100% root flooding turned black in contrast to control plants which were mostly white. Flooding reduced the concentration of potassium and calcium in petioles of 'AXR#1' rootstocks markedly. Among the four rootstocks investigated, 'AXR #1' which was the most tolerant to flooding had the best shoot and root growths, followed by 'St. George', '110R' and'420 A'.

The effect of disbudding on rooting and endogenous IAA in 'Muscat Bailey A' grapevine cuttings was investigated. Disbudding completely inhibited rooting of grapevine cuttings. More diffusible IAA was found in disbudded cuttings than in cuttings with one bud. The level of extractable IAA in the basal part where adventitious roots appear was significantly higher in the 1-bud cuttings than in disbudded cuttings 10 to 15 days after planting. It is assumed that during this period, root primordia are formed. Conversely, the applications of IBA and NAA to the disbudded cuttings induced root formation. However, the daily trends of endogenous IAA, especially that of extractable IAA, differed between the IBA and NAA treatments. Extractable IAA of disbudded cuttings treated with NAA increased for 20 days after planting, whereas in cuttings treated with IBA, there was no increase, the trend being similar to that of the control cuttings. These re-sults indicate that in the process of root formation in grapevine cuttings, a difference ex-ists between IBA and NAA action. It is also assumed that disbudding eliminated the source of extractable IAA which, therefore, inhibited rooting.

Expression and inheritance of style specific S2- and S4-allele associated S-proteins reported earlier (Hiratsuka et al., 1995) were studied using progenies of self-compatible 'Osa-Nijisseiki'.The S-protein bands of 'Osa-Nijisseiki', which was generated from self-incompatible 'Nijisseiki' as a mutant, were compared with those of 'Nijisseiki' by isoelectricfocusing polyacrylamide gel electrophoresis (IEF-PAGE). The protein band associated with S4sm-allele in 'Osa-Nijisseiki' was stained less densely with Coomassie Brilliant Blue (CBB) than did the band from 'Nijisseiki'; the RNase activity of the former band was also weaker than that of the latter as reported by Sassa et al. (1992, 1993). However, no difference was observed in the S2-protein band between 'Osa-Nijisseiki' and 'Nijisseiki'. The S-genotypes of the 'Osa-Nijisseiki' x self and 'Osa-Nijisseiki' x 'Nijisseiki' progenies were determined and their styles subjected to S-protein analyses. Progenies with the So4sm-gene from 'Osa-Nijisseiki' possessed a lightly stained S4-protein band, whereas those of 'Nijisseiki' had almost the same density as did 'Nijisseiki'. The S2-protein band was detected consistently in progenies carrying the S2-allele.Style specific S2- and S4-protein bands including a faint S4-band of 'Osa-Nijisseiki' were, thus, inherited with no exception to the progenies carrying respective S-alleles. These results may show that the proteins reported here are the S-gene products of Japanese pears and that of 'Osa-Nijisseiki' is a mutant which represses the S4-gene expression.

Three-year-old trees of some Japanese pear (Pyrus serotina Rehd.) cultivars of different growth habits (extent of apical dominance) were used. Scaffold branches were trained either vertically or laterally. Diffusible indole-3-acetic acid (IAA) levels in the terminal shoot were higher in the order of cultivars ‘Shinsui’ (strong), ‘Kosui’ (intermediate), and ‘Hosui’ (weak) in an early period, which was the reverse order of their branching ability. Scaffold branch orientation per se did not affect diffusible IAA levels in the various parts of the tree. In all cultivars, after bud break, diffusible IAA levels rapidly increased in all parts, especially in the upper part of the tree, showing a downward gradient from the shoot towards the root. A peak level in the shoot, or in the trunk, was reached prior to the period of most rapid extension growth or radial growth, respectively. The reduction of the level commenced first in the shoot, and spread basipetally towards the root. Diffusible IAA levels were associated with an increment of the growth rate rather than with the growth rate. During the growing season, extractable IAA in the cambial regions changed in a similar way to the diffusible IAA level. However, unlike diffusible IAA, extractable IAA increased and maintained a high level during the non-growing season. Removal of all shoots after cessation of the extension growth markedly decreased diffusible and extractable IAA levels in the rest of the tree.