We demonstrate an all-fiber mode locked laser based on hybrid structure of multimode fiber saturable absorber (SA) that can realize both conventional soliton and stretched-pulse states. Stable 16.44 MHz conventional soliton pulses are achieved by injecting 80 mW threshold pump power. By increasing the incident pump power to 420 mW, the laser evolves from soliton operation into stretched-pulse mode locking state. 310 fs stretched-pulse are obtained with the same repetition rate as the soliton pulses. The center wavelength and its 3 dB spectrum bandwidth are 1603 nm and 14.2 nm, respectively. For the first time, we experimentally confirm transition between conventional soliton and stretched-pulse in 1.5 μm mode-locked fiber laser by introducing multimode optical fiber SA. Moreover, the maximum single pulse energy of nearly 1 nJ is achieved. Such all-fiber mode-locked lasers based on hybrid structure of multimode fiber are attractive for practical applications without damage and the limitation of life time. In order to achieve picosecond and femtosecond optical pulses, passively mode-locking techniques based on saturable absorber are widely used. As core components, ideal SAs possessing excellent optical properties such as broad operation bandwidth, high optical damage threshold and low saturation intensity are in great demand3. Presently, the semiconductor saturable absorber mirrors (SESAMs) are widely adopted in mode-locked fiber lasers4. However, the application of SESAMs is limited due to their high-cost and limited operation bandwidths. Some two-dimensional (2D) materials based SAs have been proven to exhibit an ultrafast SA property, such as topological isulators (TIs)5, carbon nanotubes (CNT)6, black phosphorus7 and transition metal dichalcogenides (TMDs)8. These 2D materials not only possess broad operation wavelength band, but also have unique thickness-dependent bandgap. However, the gradually degraded characteristics and relatively low optical damage threshold of these SAs are the major obstacles in practical application.Ultrafast pulse fiber lasers have attracted extensive attentions due to their merits of compactness, high stability and reliability9, super continuum generation10, mode cleaning effect11, dispersive waves12, and spatiotemporal instability13. Besides, some researchers have reported mode-locked fiber lasers using the integration of MMFs14. In 2012, Mafi et al. reported ultralow-loss couplers where the nonlinear multimode interference (MMI) works15. Later, by using graded-index multimode fiber (GIMMF), Hofman et al. studied MMI based mode-field adapters in detail16. The so-called MMI is that each of excited modes periodically produces interference pattern along it when light transmits in multimode optical fiber. In the linear case, the transmission is a periodic function of GIMMF length. In the nonlinear regime, self-phase and cross-phase modulation effects could lead to the refractive index variation of excited mode when higher power transmits through the GIMMF. Therefore, the generated nonlinear phase delay will alter the interference pattern. In the year of 2013, Nazemosadat et al. theoretically researched nonlinear MMI effect and SA behavior by adopting a segment of GIMMF17. In theory, the transmission property of the SA depends on the following values: the mode number in GIMMF, the length of GIMMF, the incident pump power, the ratio of optical field diameters between the LG00 mode of GIMMF and the fundamental mode of single mode fiber (SMF). Until now, no mode-locked pulses have been experimentally generated based on this SA yet directly. Mafi notes that only when the length of a GIMMF is controlled at an odd number multiplying the beat length, could optical SA be obtained. However, self-imaging beat length of light propagating along a GIMMF is on the order of micrometers, so it is difficult to finely tune the length of a GIMMF in practice. To improve the SA based on MMF, Wang fused a segment of step-index multimode fiber (SIMMF) ahead of GIMMF in experiment18. Higher order modes of GIMMF were excited and an effective SA behavior was realized. However, the average output power was only at the level of microwatt. In 2018, Zhao et al. optimized the lengths of SIMMF and GIMMF and realized 24 mW average output pulses which results in 2.44 nJ single pulse energy19.To realize ideal SA, multimode fibers (MMFs) have been extensively studied in recent years. MMFs provide additional channels for information transmission and enable high power to transmit through. In the latest literatures, some new nonlinear phenomena and dynamics are observed in MMFs, such as multimode solitons formation7. The merit of soliton pulses is that its shape is easily maintained during transmission, therefore, the information transmitted is more accurate. Thus, these soliton fiber lasers are at the core of optical communication, sensing, optical frequency comb, nonlinear optics and so forth. For stretched-pulse fiber lasers, dispersion plays a critical role when ultrafast pulses propagate within optical fibers. By alternating normal and anomalous group velocity dispersion (GVD) in the cavity, pulses periodically experience stretching and compression. Compared with mode-locked soliton pulses, wider spectrum bandwidths and thus shorter pulse durations can be realized. These stretched-pulse fiber lasers with high output pulse energy have a wide range of applications in the domains of optical amplification, spectroscopy and optical frequency doubling. In 2014, Sotor reported the 128 fs stretched-pulse centered at 1565 nm with the help of TI20. Later, they obtained the sub-90 fs stretched-pulse by employing a graphene SA at 1.5 μm21. In 2018, Han reported conventional soliton or stretched pulses formation through employing nanotube based SA22.Additionally, it is also interesting and important to realize both conventional soliton pulses and stretched pulses mode-locked states in one laser setup. Soliton can be easily taken shape in the negative dispersion regime when anomalous dispersion interacts with and balances the nonlinear Kerr effect in the cavityIn this paper, we demonstrate a soliton-pulse evolving into stretched-pulse fiber laser by using the hybrid structure of SIMMF-GIMMF as SA for the first time. Contributed by the GIMMF, the laser can work at soliton pulse or stretched-pulse mode locking state via simply changing the pump power. Smooth stretched-pulse spectrum centered at 1603 nm with 3 dB spectral bandwidth up to 14.2 nm is observed. The obtained stretched-pulse with 310 fs pulse duration is, to the best of our knowledge, the shortest pulse generated from 1.5 μm waveband oscillators mode locked by the SA so far. The output single pulse energy can reach almost 1 nJ-level. The SIMMF-GIMMF SA exhibits high pump power tolerance and great potential for high power, large energy fiber laser generation. It is attractive to operate this high energy ultrafast soliton and stretched-pulse switchable mode locking fiber laser for a range of applications in practice.00 mode-field diameters coupling to GIMMF and the excited ones is adjustable. Therefore, the restriction on the length of the GIMMF can be eliminated. We test different lengths of SIMMF from 100 μm to 500 μm for the SA and find that it is a good strategy to get higher output power by reducing the length of SIMMF. Therefore, about 100 μm length of the SIMMF which almost reaches the limit on fiber splice is chosen in this experiment. The nonlinear transmission property is measured by using a standard 2-arm measurement system. The light source is a home-made mode-locked fiber laser . From Fig. 223, resulting in a lower mode locking threshold for laser oscillators.In this experiment, a segment of 104.4 μm SIMMF (105/125) is connected with 20 cm long GIMMF (50/125). Figure 2 fitting curve is 1.0 ps. The time bandwidth product (TBP) is calculated to be 0.416, indicating that the obtained conventional soliton pulse almost reaches transform limited state. In Fig. At first, by properly adjusting two PCs and curves of SA, self-started conventional soliton mode-locking with 16.44 MHz repetition rate can be initiated at only 80 mW pump power. Figure 14. Once the incident power exceeds 420 mW, stretched-pulse operation can be achieved instead of conventional soliton state. As shown in Fig. 19. Figure Stable soliton pulses could be kept if the injected pump power is below 390 mW. By further slightly increasing the pump power with PCs unchanged, we observe that the mode locking state becomes unstable and the fluctuation waveform of single-pulse train is obvious. This kind of multi-pulsing instability attributes to excessive non-linear phase induced by GIMMF in the mode-locked laser cavityTo evaluate stability of the oscillator in long terms, the stretched-pulse spectra are monitored and recorded every 40 minutes for total measuring time of 6 hours, as depicted in Fig. The measured optical spectra of the stretched-pulse with different length of GIMMF are sketched in Fig. Additionally, a polarization-independent isolator is chosen without initiating nonlinear polarization rotation in this experiment. To further verify the contribution of the SIMMF-GIMMF SA to mode-locking operation, the SA section is removed from the fiber laser. On this condition, no mode-locked pulses can be observed in despite of carefully adjusting the intracavity polarization state and pump power.24λ0, τ, D are resonance series, central wavelength, pulse width of soliton, dispersion parameter, respectively. Besides, DL is related to the net intracavity dispersion β2L through Eq.  is calculated to be −0.443 ps2/km and −23.25 ps2/km, respectively. Therefore, the total dispersion value is about −0.056 ps2, which facilitates the formation of conventional soliton. The output pulses are monitored via a series of devices including an optical spectrum analyzer , a digital oscilloscope (Tektronix TDS784D), a commercial autocorrelator (AInair HAC200) and a RF spectrum analyzer (Agilent technologies N9000A).The schematic of the oscillator based on SIMMF-GIMMF SA is sketched, as shown in Fig. In conclusion, we produce a hybrid structure of SIMMF-GIMMF SA with 2.85% modulation depth in our experiment. Base on this SA, a soliton and stretched-pulse switchable mode locking fiber laser is demonstrated. By raising injected pump power to 80 mW, soliton pulses with 1.0 ps pulse width are obtained at 16.44 MHz fundamental repetition rate. Conventional soliton pulses can automatically switches to stretched-pulse mode-locked state as long as injected pump power exceeds 420 mW. In this situation, the laser delivers 310 fs stretched pulses. The spectrum centering at 1603 nm with 3 dB spectral bandwidth up to 14.2 nm is achieved. For the first time, we experimentally confirm transition between conventional soliton and stretched-pulse based on the application of the hybrid structure of multimode optical fiber as SA in 1.5 μm mode-locked fiber laser. Moreover, 310 fs is the minimum pulse duration obtained based on the SA in erbium–doped fiber lasers. Besides, nearly 1 nJ single pulse energy is obtained in our experiment. These results experimentally indicate that SIMMF-GIMMF SA is feasible to form high energy femtosecond pulses.


NOG gene (c.2T>C:p.Met1?) that hinders normal translation of the noggin protein. Variants in NOG lead to a spectrum of otologic, digit, and joint abnormalities, a combination suggested to be referred to as NOG‐related‐symphalangism spectrum disorder (NOG‐SSD). Conductive hearing loss from such variants may stem from stapes footplate ankylosis, fixation of the malleoincudal joint, or fixation of the incus short process. In this case, the constellation of both stapes and incus fixation, an exceptionally tall stapes suprastructure, thickened long process of the incus, and enlarged incus body was encountered, leading to distinct challenges during otologic surgery to improve hearing thresholds. This case highlights multiple abnormalities to the ossicular chain in a patient with a start codon variant in NOG. We provide detailed imaging data on these malformations as well as surgical considerations and outcomes.We performed exome sequencing to evaluate the underlying molecular cause of a patient with bilateral conductive hearing loss due to multiple ossicular abnormalities as well as symphalangism of the fifth digits. This leads to the identification of a novel heterozygous start codon variant in the NOG gene encodes for the secreted protein noggin, which is crucial in normal bone and joint development. Noggin antagonizes bone morphogenetic proteins (BMPs) signaling, which are multifunctional growth factors that belong to the transforming growth factor beta (TGFbeta) superfamily and are important in the development of various tissues including bone and cartilage [Theartilage . de novo variants in NOG lead to a spectrum of joint/digit abnormality disorders with or without conductive hearing loss, including Type B2 brachydactyly (BDB2), multiple synostoses syndrome 1 (SYN1), stapes ankylosis with broad thumbs and toes (Teunissen–Cremers syndrome), proximal symphalangism 1A (SYM1A), and tarsal-carpal coalition syndrome (TCC). Overall, this can be referred to as NOG‐related‐symphalangism spectrum disorder (NOG‐SSD) in a single unified diagnostic term [Autosomal dominantly inherited ortic term . NOG gene causing NOG symphalangism syndrome (NOG-SSD) including incus malformations and report its clinical and molecular phenotype as well as surgical considerations and results for the patient's conductive hearing loss.Herein, we describe a novel variant in theAfter IRB approval (H-17566) at the Baylor College of Medicine, the patient's complete medical and family history was reviewed with attention to symphalangism and conductive hearing loss. Study participants provided written, informed consent. Pre- and postoperative audiometry included pure tone audiometry and tympanometry, as well as ipsilateral and contralateral stapedial reflex testing. High-resolution computed tomography (CT) of the temporal bone without contrast was performed utilizing axial slices with coronal and sagittal reconstructions to evaluate the middle and inner ears. NOG. In short, purified PCR products  were sequenced using the BigDye Terminator v3.1 Cycle Sequencing Kit followed by capillary electrophoresis on an ABI 3730 DNA Analyzer .A saliva sample was obtained from the patient using the Oragene OG-500 collection kit and DNA was extracted via the prepIT purification kit . Exomic libraries were prepared using the SureSelect Human All Exon V6 kit, and sequencing was performed on a HiSeq 2500/4000) instrument. Reads were aligned to the human reference genome using Burrows-Wheeler Aligner BWAv0.7.15) [000 instr.15 [000 Our patient is a 35-year-old Caucasian male referred for bilateral progressive conductive hearing loss since early childhood. Evaluation at the age of ten included audiometry but no imaging. Reportedly, this demonstrated some degree of hearing loss but was not available for review. Medical history included an abdominal lipoma, open heart surgery for aortic valve anomaly at the age of 5, and multiple orthopedic surgeries including excision of a ganglion cyst of the wrist, as well as repair of shoulder and ankle fractures from injury-appropriate sports-related trauma not thought to be related to his phenotype. Notably, the patient denied ophthalmologic history. He had a family history of hearing loss in his father and paternal grandfather. Reportedly, his grandfather had similar symphalangism of both fifth digits, while his father did not. Neither were available for genetic testing or examination. Physical examination of the hands demonstrated symphalangism of both fifth digits only. He did not have proximal symphalangism of the other digits or noted brachydactyly. Imaging of the hands was not performed at our institution due to prior work-up with his primary physicians. Binocular microscopy demonstrated bilateral normal tympanic membranes and well-aerated middle ear spaces, and pneumatic otoscopy displayed mobility of the malleus bilaterally. Pure tone audiometry revealed bilateral moderate rising to mild conductive hearing loss bilaterally . Speech After appropriate counselling, the patient underwent surgical management to address his right conductive hearing loss. Intraoperatively, ossicular abnormalities including an enlarged, fixed incus as well as fixed stapes were confirmed. The incus was mildly fixed and was mobilized using gentle traction between the body and the scutum. The patient underwent right stapedotomy with CO2 laser and reconstruction with a 5 mm Richards piston over fascia seal of the oval window. His postoperative course was unremarkable. The patient's postoperative pure tone and speech recognition thresholds improved significantly after surgery . He unde NOG gene in the patient (NM_005450: c.2T>C:p.Met1?), which was verified by Sanger sequencing. This variant is absent from the gnomAD database and is predicted to be deleterious by various bioinformatic tools included in dbnsfp33a  [Exome sequencing revealed a heterozygous start codon variant in theM, SIFT) . The varM, SIFT) . NOG variants, conductive hearing loss has mainly been described as a result from stapes ankylosis. However, other ossicular chain abnormalities have also been observed in a few cases, such as fixation of the malleoincudal joint, fixation of the short process of the incus, and elongation of the long process of the incus [ NOG variants. Thus, we feel CT imaging is useful in preoperative planning to anticipate difficulties with prosthesis placement.In patients withhe incus . Preoper NOG variants [Variable hearing results are reported after stapedectomy forvariants –11. Browvariants . All autvariants . NOG gene (c.2T>C:p.Met1?) in a patient with bilateral progressive conductive hearing and symphalangism in the fifth digits. This variant leads to a loss of the translation start site of the NOG gene and is predicted to lead to a complete loss of function of the noggin protein. We highlight that multiple malformations in the middle ear ossicular chain can exist with NOG variants.In conclusion, exome sequencing was utilized to identify a novel variant in the


Firefighters are susceptible to auditory dysfunction due to long-term exposure to noise from sirens, air horns, equipment, and tools used in forcible entry, ventilation, and extrication. In addition, they are exposed to ototoxic chemicals, particularly, during overhaul operations. Studies indicate that 40% of firefighters have hearing loss in the noise-sensitive frequencies of 4 and 6 kHz. Noise-induced hearing loss (NIHL) is often accompanied by tinnitus, which is characterized by ringing noise in the ears. The presence of phantom sounds can adversely affect the performance of firefighters. However, there has been limited research conducted on the prevalence of tinnitus in firefighters. We enrolled firefighters from Michigan, with at least 5 years of continuous service. The hearing handicap inventory for adults (HHIA) was used to determine the difficulty in hearing perceived by the firefighters and the tinnitus functional index (TFI) was used to determine the severity of tinnitus. Self-perceived hearing handicap was reported by 36% of the participants, while tinnitus was reported by 48% of the participants. The TFI survey indicated that 31% perceived tinnitus as a problem. More importantly, self-perceived hearing handicap was significantly associated with the incidence of tinnitus in firefighters, suggesting a potential link between occupational exposure to ototraumatic agents and tinnitus in firefighters. Hearing loss, the third-most common chronic physical condition in American adults, can significantly affect quality of life and productivity . ApproxiTinnitus has been reported to often accompany sensory neural hearing loss induced by noise exposure, as well as aging. In the absence of normal auditory signals from the cochlea, the central auditory system undergoes abnormal neuroplastic changes, which may downregulate centrally-mediated inhibition and cause tinnitus . EnhanceUrban areas, such as Detroit, were heavily industrialized in the past and have an older housing stock. A recent study indicated that the levels of lead and cadmium are at much higher concentrations in southeast Michigan, probably due to leaking and spillage of chemicals from various industries . ConsequA cross-sectional design was used for this exploratory study, which was approved by the Wayne State University’s Institutional Research Board (IRB # 064814MP2E & 084017MP2F). Firefighters were recruited from several engine houses in Michigan. Invitations to participate in this study were given through the Fire Department, as well as through the Fire Fighters Association. Research personnel also visited engine houses to introduce the study. Firefighters expressed their interest in participating in the study via telephone, email, or in-person during engine house visits. Three different survey forms were distributed to the recruited participants to determine prior exposure to ototraumatic agents, self-perceived hearing handicap, and incidence of tinnitus. The questionnaires were completed by firefighters at the engine houses or in the clinic, in the presence of research personnel, so that any clarification needed for answering the questions was provided immediately. Career firefighters, who have worked for a minimum of 5 continuous years, were included in the study, while those with a familial history of hearing loss, middle ear disease, other serious ailments, or large differences in the hearing threshold (>40 dB) between left and right ear were excluded from the study. Pure-tone audiometry was used to identify large differences in hearing thresholds. Written consent was obtained prior to participation in the study.Potential exposure to ototraumatic agents and environmental factors that influence hearing was assessed using a “History of Prior Exposures” questionnaire. In addition to demographic information, this questionnaire was used to obtain information regarding the number of years in service as a firefighter, job title, exposure to non–work noise , use of medications, serious illness , injuries to ear or head, family history of hearing problems, use of implants or hearing aids, other ear problems , exposure to solvents or vibration, and habits . This information was used to identify firefighters who have been exposed to multiple risk factors so that they could be excluded from the analysis.Self-perceived hearing handicap in firefighters was assessed using the HHIA survey . This isThe TFI questionnaire, a 25-item self-reported instrument developed by Meikle et al (2012), was used to determine the presence of tinnitus. This survey includes 8 subscales: Intrusive, sense of control, cognitive, sleep, auditory, relaxation, quality of life, and emotional to characterize the severity of tinnitus and assess its impact. Each subscale contains 3 questions, whereas quality of life subscale contains 4 questions. The total score ranges from 0 to 100. This survey has been demonstrated to have an excellent test-retest reliability of 0.78 and high internal consistency reliability with a Cronbach’s alpha value of 0.97 . The TFIp-value of <0.05 was considered to indicate a statistically significant association between the variables. The results are expressed as mean ± standard deviation. Linear regression analyses were used to determine the relationships between age, number of years in service as a firefighter, self-perceived hearing handicap (HHIA score), and the severity of tinnitus (TFI score). A bivariate regression analysis was employed to determine the association of: (1) age with HHIA and TFI scores; (2) number of years in service with HHIA and TFI scores; and (3) HHIA with TFI scores, precluding the necessity to correct for multiple comparisons. The data was analyzed in Microsoft Excel using Data Analysis Toolkit . A Demographic characteristics of firefighters in our study are shown in Self-perceived hearing handicap, assessed using the HHIA scores, indicated that 64% of firefighters had no handicap, 29% had a moderate handicap, and 7% had a significant handicap . Scores The presence of tinnitus was determined using the “History of Prior Exposure” survey, which specifically inquired whether the firefighter has a ringing noise in the ear (Question #7). The severity of tinnitus was assessed using the TFI questionnaire. Among the 42 firefighters who participated in this study 52% did not report tinnitus, 17% did not perceive tinnitus as a problem, 9% perceived tinnitus as a mild problem, 17% perceived tinnitus as a mild to moderate problem, and 5% perceived tinnitus as a moderate problem or greater . Scores p-value = 0.25 and 0.07, respectively). Though the number of years in service was not associated with tinnitus  it was associated with hearing handicap (p-value = 0.004; p-value = 0.003; We examined the association between different variables by using linear regression analysis. Age and years in service were considered as independent variables while tinnitus and hearing handicap were considered as dependent variables. Age was not associated with the presence of tinnitus and hearing handicap (Firefighting is a hazardous job with significantly high risk of work-related injuries. Good hearing is essential for firefighters to take appropriate and timely action in critical situations, and tinnitus has the potential to interfere with their life-saving actions. In this study a cohort of 42 firefighters from the Detroit metropolitan area was recruited to assess the incidence of tinnitus. Results show that a third of the firefighters have some degree of difficulty in hearing and that 31% have tinnitus with different degrees of severity. In addition, we found a statistically significant association between years in service and hearing handicap and between hearing handicap and tinnitus. Considering that tinnitus is found in 10–15% of adults , our resSubjective tinnitus is characterized by the perception of phantom sound, without external acoustic stimuli, that can be heard only by the afflicted person. It affects between 10% and 15% of the adult population all over the United States and Europe. It is stressful and very damaging to the overall health of patients. Those who have tinnitus complain of various symptoms commonly associated with the condition, such as sleep deprivation, difficulty in working, depression, anxiety, and various other health impairments ,14. A stAlong with loud noise exposure, firefighters may be exposed to multiple ototraumatic pollutants every day. Heavy metal exposures, with or without noise overexposure, are most frequently associated with hearing loss and tinnitus ,16. A stThe strength of this study is that it focuses on a specific population that is continuously exposed to multiple ototraumatic agents that are commonly found in urban communities. However, there are some limitations that should be considered while interpreting the findings of this study. First, the number of participants was relatively low, as the total size of our target population is small. Second, because the participation in this study was voluntary, the sample may be biased. For example, firefighters with considerable hearing difficulties may not be inclined to reveal their health issues and may not have volunteered. Third, although African American firefighters represented about 36% of the participants, this study lacks representation from other minority groups, such as Hispanics, Asians, and Native Americans. Finally, the prior exposures survey is prone to recall bias and some data collected in this study may be subjective, as it is based on a self-assessment of difficulties in hearing and problems associated with tinnitus. Nevertheless, the findings of this study could be extrapolated to those who are exposed to multiple ototraumatic agents in other occupational settings and urban localities.Firefighters are at risk of hearing loss and tinnitus, probably due to increased exposure to multiple ototraumatic agents in the firefighting environment. The significant associations between number of years in service and hearing handicap, and between hearing handicap and tinnitus, highlight the importance of preventive measures in limiting exposure to occupational risk factors. The findings of this study would be useful for directing efforts to advocate for better occupational health policy and minimize the incidence of hearing loss and tinnitus in firefighters.


Triclofos sodium (TCS) and chloral hydrate (CH) are widely used as sedatives for children, but no analytical method to simultaneously monitor concentrations of blood TCS, CH and their metabolites, trichloroacetic acid (TCA) and trichloroethanol (TCEOH), has been reported. The present study aimed to develop a simple analytical method for TCS and its metabolites  in small-volume plasma from children. After acidification of specimens, TCS formic acid adduct or the metabolites derivatized using water/sulfuric acid/methanol  were measured by combined use of liquid chromatography tandem-mass spectrometry and gas chromatography mass-spectrometry. The limits of detection and quantification levels (µg/ml) were 0.10 and 0.29 for TCS, 0.24 and 0.72 for TCA, 0.10 and 0.31 for TCEOH, and 0.25 and 0.76 for CH, respectively. The mean recoveries were 82.8–107% for TCS, 85.4–101% for TCA, 91.6–107% for TCEOH, and 88.9–109% for CH. Within-run and between-run precision  using this method ranged from 1.1 to 15.7% and 3.6 to 13.5%, respectively, for TCS and all of its metabolites. The calibration curves were obtained with standard spiked plasma, and all of the coefficients of determination were more than 0.975. Subsequently, we applied the present method to plasma taken from five children after sedation induced by CH and TCS. In addition to TCS and CH, elevated TCA and TCEOH concentrations were detected. This new method can be applied for the pharmacokinetic analysis of TCS and its metabolites and the determination of the optimal TCS dosage in children. TCS and CH are allowed to be used as oral or intra-rectal agents to safely induce hypnosis in pediatric patients. In Japan, these sedatives are usually used in the range from 20 to 80 mg/kg for TCS and 30 to 50 mg/kg for CH. However, adverse effects have been reported, including cardiac arrhythmia4, respiratory insufficiency and esophagitis5 after overdosing . Appropriate dosages of TCS and CH are needed for safer sedative protocols.Triclofos sodium (TCS) and chloral hydrate (CH) are widely used for procedural sedation for diagnostic imaging studies and for prolonged sedation in pediatric intensive care unit7. CH is reversibly metabolized to TCEOH by alcohol dehydrogenase (ADH)8. Aldehyde dehydrogenase (ALDH) oxidizes CH to trichloroacetic acid (TCA), which is excreted into the urine9 and the bile10 as TCA glucuronide. The blood TCEOH level rapidly increases after TCS or CH administration and is eliminated from plasma with a half-life (t1/2) of 8.2 hours. In contrast, the plasma TCA level increases slowly and the t1/2 is 75.3 hours11. Further pharmacokinetic analyses of TCEOH and TCA are necessary in the pediatric population because children less than six months old are at higher risk of adverse events after administration of CH than older children12. However, there have been no reports on methods for simultaneously monitoring TCS and its metabolites in the blood.TCS is rapidly hydrolyzed to trichloroethanol (TCEOH), which contributes to the sedation, and monosodium phosphate Fig.  after adIn this study, we aimed to develop and validate a new, simple analytical method to determine TCS and its metabolites in the plasma after sedation is induced, using the combination of liquid chromatography tandem-mass spectrometry (LC-MS/MS) and gas chromatography-mass spectrometry (GC-MS).Prior to sample collection from pediatric patients, written informed consent was obtained from their parents as legal guardians in the University Hospital, Kyoto Prefectural University of Medicine. The protocol of this study was reviewed and approved by the medical ethics committee of the Kyoto Prefectural University of Medicine and the ethical review committee of the Nagoya City University Graduate School of Medical Sciences . This study was carried out in accordance with the Helsinki Declaration, and the Ethical Guidelines for Medical and Health Research Involving Human Subjects established by the Ministry of Education, Culture, Sports, Science and Technology and the Ministry of Health, Labour and Welfare in Japan. The inclusion criteria of the participants were: (i) under 12 years old with body weights >2 kg; (ii) receiving treatment in the pediatric intensive care unit for 3 days or more; and (iii) having a catheter placed in the central vein or artery for treatment purposes, which enabled non-invasive blood sampling. Patients with (i) allergy to TCS or/and CH, or (ii) cardiovascular collapse due to hemorrhage or severe cardiac dysfunction were excluded.2), used as an internal standard (IS), and TCEOH were obtained from Sigma-Aldrich  and triclofos sodium (Tricloryl® syrup 10%) was from Alfresa Pharma Corporation . Methanol (LC/MS grade), ultra-pure water (LC/MS grade), hexane (high-performance liquid chromatography (HPLC) grade), ethyl acetate (pesticide residue grade), sulfuric acid , formic acid (LC/MS grade), and ammonium formate solution  were purchased from Wako Pure Chemical Industries. Sulfatase from Helix pomatia (type H-2), including glucuronidase activity, was obtained from Sigma-Aldrich.TCA (molecular biology grade) and CH (1st grade) were purchased from Wako Pure Chemical Industries . Dichloroacetic acid di-deuterium . The pooled plasma from three healthy volunteers (written informed consent was obtained), who were neither treated with any drugs nor exposed to chemicals before the sampling, were spiked with standard solutions and used for the basic methodological examination in this study.6. Twenty-five microliters of the spiked plasma were pipetted into an Eppendorf tube, and 10 µl of sulfuric acid (0.1 mol/l) and 25 µl of DCA-d2 (1 mg/ml in methanol) were added. After vortex, 100 µl water/sulfuric acid (0.1 mol/l)/methanol  were added to the sample mixture and incubated at 70 °C for 10 min. Afterwards, 110 µl of n-hexane/ethyl acetate  were added, and the mixture was shaken vigorously for 20 min and centrifuged (4000 × g for 10 min at 25 °C). The organic solvent layer was transferred to vials and then used for the analyses of TCS by LC-MS/MS and the analyses of the metabolites  by GC-MS .The procedure for determining plasma TCS and its metabolites is shown in Fig. ® HSS T3 column  with 2.1 × 100 mm i.d. and 1.8 µm silica. Mobile phases were 10 mmol/l ammonium formate and 0.2% formic acid , and 10 mmol/l ammonium formate in methanol (B). Total flow rate of the mobile phase was 0.2 ml/min. Total run time including equilibration was 10 minutes. The initial mobile phase composition was 95% mobile phase A and 5% mobile phase B. The percent of mobile phase B was increased linearly over the next 5 minutes to 80%, held for 1 minute, and then increased linearly up to 95%. After that, the percentage was held for 1 minute, and the mobile phase composition returned to the initial condition where it was held for 2 minutes. The injection volume was 10 µl. The MS/MS was operated with an electrospray ionization source in the negative ion mode with multiple reaction mode. The nebulizer nitrogen gas flow was set at 1.5 l/min with the source temperature of 350 °C and drying nitrogen gas flow at 15.0 l/min. The temperatures of the desolvation line and heat block were 150 °C and 100 °C, respectively.LC-MS/MS analysis of TCS was run on a LC-MS/MS-8030 system  composed of a solvent delivery device (LC30AD), an auto sampler (SIL-30AC), a system controller (CBM-20A) and a column thermostat (CTO-20A). The LC-MS/MS conditions were as follows. The LC column was an ACQUITY UPLC2 (IS) and TCA, m/z 49 for TCEOH and m/z 61 for CH . The GC operating conditions were as follows. The GC column was Rtx-65  with 30 × 0.25 mm i.d. and 0.25 µm film thickness. Initial column temperature was 50 °C, elevated to 95 °C at 5 °C/min, and then to 240 °C (held for 2 min) at 75 °C/min. The injection port temperature was 150 °C. Helium (99.999% purity) carrier gas was used with a flow rate of 1 ml/min. The injection volume was 1 µl (splitless). The temperatures of the MSD transfer line, ion source and quadrupole were 280, 230 and 150 °C, respectively. The analysis used electron ionization with 70 eV in positive ion mode. The selected ion monitoring parameters were m/z 59 for DCA-dAnalyses of TCS and the metabolites were carried out with an external standard method and an isotope dilution method, respectively. The linearity of the calibration curve, using pooled plasma spiked with standard solutions of TCS and its metabolites at final concentrations ranging from 0.5 to 150 or 300 (for TCEOH only) µg/ml (six or seven points), was determined by linear regression analysis. Since the concentration range of 0.5 to 75 µg/ml for TCS, TCA and CH was found to sufficiently cover the clinical concentration range and the slopes of the calibration curves were almost the same using either six or five concentration levels (data not shown), we used calibration curves derived from five levels (from 0.5 to 75) for routine analyses of these compounds. Separate calibration curves with different slopes were used to quantify high  and low  concentrations.13: LOD = t  × s; where t = Student’s t (one-sided), n-1 = degrees of freedom, and s = the standard deviation of seven replicates at the lowest concentration level of the calibration curve corresponding to a signal-to-noise ratio of three. The limit of quantifications (LOQ) was defined as 3 × LOD.The limit of detection (LOD) levels were calculated from the following equationsThe within-run accuracy and precision were evaluated by spiking standards into the pooled plasma at concentrations of 0.5, 5, 25, 75, and 300 µg/ml for TCEOH and 0.5, 5, 25, and 75 µg/ml for the other analytes (n = 7). In addition, the between-run accuracy and precision were examined on seven different days with seven assay runs of the pooled plasma spiked with standards at the same concentrations adopted in the within-run assays. The percent of the nominal concentration was calculated to determine the accuracy, and the relative standard deviation (%RSD) was calculated to determine the precision.For stability, the analyte concentration in the matrix was determined using spiked plasma samples at two concentrations . Plasma samples were incubated at 4 and 37 °C for 24 hours, after which TCS and its metabolites were measured. The accuracies of plasma samples incubated for 24 hours were compared with those of plasma before incubation. The stability of prepared samples injected into the GC-MS and LC-MS/MS was also tested at the same concentrations. The analytes in prepared samples were analyzed immediately and after storage for 4, 8 and 24 hours in each GC-MS (at room temperature) and LC-MS/MS (at 4 °C) autosampler. Stability was assessed by comparing the peak areas of samples at each time point within 24 hours with those of samples stored for 0 hours. Additionally, plasma samples prepared at 0 °C were compared with those prepared at room temperature.To evaluate the matrix effect, three individual plasma samples at four concentrations  were prepared and analyzed by the procedure in this study (n = 3). Carry-over in the blank sample was assessed by injecting blank samples after measuring samples with analytes at high concentrations (150 or 300 µg/ml).To examine whether the newly developed method could be used to monitor TCS and its metabolite concentrations in children’s blood, it was applied to 16 plasma samples taken from five children  who were under sedation following operations for congenital heart defects. Post-operative sedation in the pediatric intensive care unit was managed by the rectal administration of CH (31–49 mg/kg), followed by oral administration of TCS (61–79 mg/kg). Blood was collected three times . In one participant to whom TCS was administered five times, an additional blood sample was taken before the first TCS administration. The plasma samples were obtained by centrifugation and stored at minus 80 °C until the assay.14, it was reported that sulfate conjugation activities for some drugs are more active than glucuronidation in neonates and infants16. It is thus possible that conjugated-TCEOH might have included both glucuronide and sulfate in the children’s samples. Hence, we decided to use sulfatase for conjugate dissociation. The sulfatase (61 units) included 21844 units of β-glucuronidase as well because sulfatase from Helix pomatia has secondary activity of glucuronidase17.In order to detect conjugated TCEOH in the plasma, the samples were hydrolyzed by adding sulfatase (61 units) and incubating for 40 hours, which was the optimal condition determined in this study (see Results and discussion section). Although glucuronide of TCEOH is a well-known metabolite, and a sulfate conjugate has not been documented in previous studies conducted mostly among adults18.When the concentrations were below the respective LODs, the values of LOD/square root of 2 were assigned for further calculation. Statistical analyses were conducted with EZR version 1.32 for WindowsWritten informed consent was obtained from guardians of the children participants in the University Hospital, Kyoto Prefectural University of Medicine. This study was approved by the medical ethics committee of the Kyoto Prefectural University of Medicine and the ethical review committee of the Nagoya City University Graduate School of Medical Sciences .21. The TCS adduct with formic acid, a precursor ion of TCS, showed the most effective transition to the product ions, whereas the other adducts such as acetic acid did not show effective transition (data not shown).The optimized parameters and retention times of analytes are shown in Table The LOD, LOQ, recovery, precision and stability are summarized in Table The stabilities of TCS and its metabolites in plasma and the prepared samples were assessed under typical storage/handling conditions. Incubation of plasma samples at 4 or 37 °C caused negligible changes in accuracy for TCA, although other analytes showed a decrement in accuracy of more than 15%. These results suggest that prompt freezing after sampling and immediate preparation after thawing are needed. TCS in plasma in particular was rapidly hydrolyzed at high concentration, although we ascertained that the preparation at 0 °C (on ice) showed approximately the same level as the nominal concentrations (data not shown). A time course analysis of the prepared samples revealed that the results measured within 24 hours were not different from those measured immediately after the preparation, except for TCS (at 75 µg/ml). However, since this analytical method was reliable for the low concentration of TCS, it was thought that TCS of the clinical samples in this study were well quantified because the maximum concentration of TCS was approximately 5 µg/ml.The average %RSD of the matrix effect within the concentration range of 0.5–75 µg/ml was 13.9% for TCS, 5.6% for TCA, 8.9% for TCEOH and 14.9% for CH (detailed data not shown). In a carry over test, no analytes were detected in the blank sample.22. Reported LOD values of TCEOH and TCA in human plasma are 0.12 and 0.22 µg/ml6, and 0.06 and 0.04 µg/ml19, respectively. For CH, the LOD in rat and mouse sera has been reported to be 0.2 µg/ml23. TCEOH concentrations in the blood after single administration of TCS (22 mg/kg) and CH (15 mg/kg) were reportedly 8.2 and 8.5 µg/ml, respectively11. Since both sedatives are usually used in the range from 20 to 80 mg/kg for TCS and 30 to 50 mg/kg for CH in Japan, we consider that the present method may make it possible to analyze the plasma concentrations of TCS and its metabolites  in the course of sedative treatment.The present study successfully quantified blood TCS concentrations for the first time. The LODs of the three TCS metabolites, which were measured along with TCS, were in orders similar to those reported in a previous studySince the hydrolyzing procedure using sulfatase should be optimized, free TCEOH concentrations yielded after different procedures were investigated. Sixteen-hour incubation Fig.  was not The sulfatase included 21844 units of β-glucuronidase. Deconjugation achieved with 224 units of commercial β-glucuronidase obtained from Sigma-Aldrich  was equivalent to that achieved with the above sulfatase under incubation for 40 hours (data not shown). Though it was not clear whether TCEOH sulfate existed in the blood, this optimized method using the sulfatase was considered to hydrolyze both glucuronide and sulfate conjugates . Based on the above results, we determined the optimum hydrolyzing condition to be a 40-hour incubation at 37 °C using 61 units of the sulfatase.11. The child in Fig. The measurement results of TCS and the metabolites in plasma taken over time from five children under sedation in a pediatric department are shown in Fig. 24 after 40 mg/kg dosing and CH was almost cleared from children’s plasma between 15 and 30 min after a single administration of 50 mg/kg8. In the present study, multiple dosing might have resulted in the extended biological half-life. CH was also not detected in one patient to whom CH was not administered  samples, and CH was detected in 8 out of 16 plasma (50.0%) taken from five children Fig. . AccordiAn appropriate method is required for small-volume blood samples for analyses of TCS and its metabolites in young children. The 25 µl of plasma needed for the present procedure meets this requirement. Additionally, the present method of sample preparation is simple and allows injection of the same pretreated specimens into LC-MS/MS and GC-MS. Only 4 hours or less are needed for sample preparation, and the necessary amount of organic solvent can be saved through the operation of analysis. Altogether, the advantages of this newly established method are the low sample volume needed and simple preparation for measurements of TCS and its metabolites.The present study succeeded in developing a reliable and simple method of detecting TCS and its metabolites in blood. This method can be applied to routine examinations for pharmacokinetic analyses of TCEOH and TCA after TCS treatment.


Tinnitus is a common symptom among patients with hearing loss, and many studies have reported successful tinnitus suppression with hearing devices. Active middle ear implantation of the Vibrant Soundbridge (VSB) is a good alternative to existing hearing devices. This study evaluated the effects of VSB implantation on tinnitus and sought to identify the main audiological factor that affects tinnitus suppression.The study participants were 16 adults who had tinnitus with sensorineural hearing loss, and who underwent VSB implantations. Pure-tone audiometry; word recognition test; tinnitus handicap inventory (THI); and visual analog scale (VAS) assessment of loudness, awareness, and annoyance were performed before and 12 months after surgery. Changes in hearing threshold, word recognition scores (WRS), THI scores, and VAS scores were analyzed.VAS scores for loudness , awareness , and annoyance  showed significant improvements from baseline to 12 months after surgery. In addition, THI scores showed a significant decrease . The average hearing threshold level, WRS, and most comfortable level (MCL) also showed significant improvements at 12 months after surgery . Among the aforementioned factors, changes in MCL were best correlated with those in THI scores .A VSB implant is beneficial to subjects with tinnitus accompanied by sensorineural hearing loss. The changes in THI scores best correlated with those in MCL. This improvement may represent a masking effect that contributes to tinnitus suppression in patients with VSB implants. Tinnitus is the perception of sound in the absence of sound stimuli, and affects approximately 10–15% of the adult population . TraditiMany studies have reported tinnitus suppression following auditory stimulation with hearing devices, such as hearing aids (HAs), cochlear implants, and more recently, middle ear implants. As early as 1947, Saltzman and Ersner reported that patients with tinnitus benefited from the use of HAs , and theSeveral possible mechanisms can explain tinnitus suppression by means of hearing devices. One possible mechanism involves hearing gain. Recent neuroimaging studies have revealed that tinnitus brain networks include not only sensory auditory areas, but also cortical regions involved in perceptual, emotional, memory, attentional, and salient functions , 16. EmoThe Vibrant Soundbridge  is a partially implanted, active middle ear device. It directly delivers mechanical vibrations to the middle ear by placing the floating mass transducer (FMT) on the middle ear structures. Previous studies have confirmed that it confers superior, more usable amplification, as well as easier communication in daily listening environments than conventional amplification via HAs . AccordiThe purpose of the present study was to investigate the effects of VSB on tinnitus and to identify the main audiological factor involved in the changes in tinnitus, with a view to improving the treatment of tinnitus in patients with sensorineural hearing loss.This study involved a retrospective chart review of patients who had sensorineural hearing loss with tinnitus, and who underwent VSB implantation at the Severance Hospital in Seoul, Korea, during January 2016 to July 2019. Inclusion criteria were 1) sensorineural hearing loss accompanied by ipsilateral tinnitus, 2) VSB implantations performed in the affected ears, 3) stable chronic tinnitus that did not respond to any previous treatments, and 4) follow-up for more than 1 year after VSB implantation and completion of audiological tests and self-assessment questionnaires preoperatively and at 1 year postoperatively. Exclusion criteria were 1) chronically disabled middle ears or previous ear surgeries, 2) fluctuating hearing and/or tinnitus, and 3) known conditions that could cause hearing loss and/or tinnitus .In total, 47 patients underwent VSB implantation between January 2016 and July 2019. From these, 3 patients were excluded because they had mixed hearing loss. In addition, 16 patients who did not experience tinnitus, 4 who were followed up for less than 1 year, 7 who did not complete the questionnaires, and 1 foreigner for whom a speech discrimination test was not available were excluded. Finally, 16 subjects  were enrolled in this study. The mean age at operation of the 16 patients was 66.0 ± 8.2 (53–76) years. All patients had moderate to severe sensorineural hearing loss (58.9 ± 6.9 dB HL) accompanied by subjective tinnitus on the same side. Hearing loss was bilaterally symmetric in 12 patients, and asymmetric in 4 patients. Those 4 patients had not recovered from previous sudden unilateral hearing loss, and the implantations were performed in the worse ears. The configuration of hearing loss in the implanted ears was flat in most patients , followed by sloping  and ascending . The patients’ demographic data are listed in All procedures were performed in accordance with the 1975 Declaration of Helsinki. The study was approved by the institutional review board of the Severance Hospital in Seoul, Korea (4-2017-0847). All of the subjects provided informed consent to participate in the study.All patients were assessed by using pure-tone audiometry and word recognition tests  at preoperative and 12-months postoperative time points. The pure-tone air (250–8000 Hz) and bone conduction (250–4000 Hz) thresholds were measured using clinical audiometers in a double-walled audio booth. The pure-tone average (PTA) was defined as the mean value of the measurements taken at frequencies of 500 Hz, 1 kHz, 2 kHz, and 4 kHz. A word recognition test was performed to obtain the maximal WRS, which was measured at the most comfortable hearing level (MCL) using 50 monosyllabic Korean words that are commonly heard during everyday life. The Korean words were from a validated and standardized resource  and werePatients were reassessed 12 months after surgery, and the assessments consisted of aided pure-tone audiometry and a word recognition test. Aided pure-tone audiometry was performed after switching on the VSB alone using sound field (air conduction) testing through speakers with contralateral masking noise, which was achieved via a calibrated headphone. The masking procedure was the plateau method, also known as Hood’s technique .Functional hearing gain (FHG) was determined as the difference between preoperative and postoperative free-field audiometry data.To assess pre-operative and postoperative tinnitus severity, all patients completed 2 self-administered questionnaires: a visual analog scale (VAS) for loudness, awareness, and annoyance, with ratings from 0 to 10, and the tinnitus handicap inventory (THI) developed by Newman et al. . The quet-test or the paired t-test for evaluating differences between 2 groups if the data exhibited a normal distribution. Multiple regression analysis was performed to identify which audiological factors affected the improvement of tinnitus using a general linear model. Values of p < 0.05 were considered to be statistically significant.Statistical analysis was performed with SPSS for Windows, version 21 . The results of multiple experiments are presented as the mean ± standard deviation. Comparisons were performed between continuous variables using Student’s t = 2.7, p < 0.05). Considering the criterion of a 20% decrease in THI score, 7 patients (43.8%) reported improvement of tinnitus; however, 8 patients (50.0%) reported no change, and 1 patient (6.2%) reported worsening of tinnitus.The therapeutic effects of VSB on tinnitus were encouraging . The init = 3.3, p < 0.01). VAS scores for awareness and annoyance also decreased from 6.3 ± 2.1 to 4.7 ± 1.9  and from 6.3 ± 2.0 to 4.6 ± 1.8 , respectively. Based on the 20% criterion, 7 patients (43.8%) reported improvement in tinnitus loudness, 8 patients (50.0%) reported no change, and 1 patient (6.2%) reported louder tinnitus after implantation. The proportions were similar in the other 2 questionnaires. For awareness, 8 patients (50.0%) reported improvement, 6 patients (37.5%) found no change, and 2 patients (12.5%) reported worsening. For annoyance, 8 patients (50.0%) reported improvement, while 7 (43.8%) were stable and 1 (6.2%) was more annoyed after implantation than under the unaided condition  self-reported questionnaire. The questionnaire results demonstrated statistically significant improvements after VSB implantation. VAS scores for loudness decreased from 6.8 ± 2.3 to 4.9 ± 1.5 , and the FHG was 17.3 ± 11.9 dB. Word recognition tests were performed under the preoperative unaided condition, and under the VSB-aided condition at 12-months postoperatively. The patients’ WRS improved from 62.1 ± 10.6% to 69.8 ± 13.9% , and their MCL decreased from 83.5 ± 5.9 dB to 57.3 ± 3.5 dB  average FHG, 2) changes in MCL, and 3) changes in WRS. The improvement of tinnitus was measured as 1) changes in THI, 2) changes in VAS for loudness, 3) changes in VAS for awareness, and 4) changes in VAS for annoyance. When we performed multiple regression analysis, only decreased MCL was significantly and positively related to the improvement of THI. Improvement of MCL (unstandardized regression coefficient = 2.55 with standard error = 0.76) explained 60.0% of the THI improvement Click here for additional data file. 19 Aug 2019Attachmentrebuttal_letter.docxSubmitted filename: Click here for additional data file. 20 Nov 2019PONE-D-19-23345Effects of Vibrant Soundbridge on tinnitus accompanied with sensorineural hearing lossPLOS ONEDear Professor Moon,Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.We would appreciate receiving your revised manuscript by Jan 04 2020 11:59PM. When you are ready to submit your revision, log on to If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocolsTo enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. 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Please see our Supporting Information guidelines for more information: Additional Editor Comments:I have reviewed the manuscript in addition to an independent reviewer with expertise in this area and also a statistical advisor. We think the manuscript require some revision before considering for publication.[Note: HTML markup is below. Please do not edit.]Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: NoReviewer #2: Yes**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: NoReviewer #2: Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: NoReviewer #2: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: 1. Page 2, lines 24-29: Instead of showing p-values, I suggest to depict point estimates and their 95% confidence intervals.2. Page 5, lines 91-93: I was curious how many subjects were not included because of the lack of follow-up, audiological tests, and/or self-assessment questionnaires. Inclusion criteria #1-#3 and exclusion criteria #1-#3 would provide the number of study subjects within a 7-month period.3. Table 2:(3a) Column "A-B": I assumed the test for A-B comparison was a paired t test. For paired t tests, I would suggest to replace the percent change with a standard deviation of the difference. The percent change can be easily calculated by readers if necessary.(3b) Column "Probability": I suggest to replace "probability" with 'p-value'.4. Page 10, lines 189-198: With the sample size of 16, the regression analyses were likely to be under power, meaning that the only significant finding might not be reproducible  or falsely observed . Please discuss this as a limitation in the Discussion section.5. Table 3: I suggest to replace the standard errors with 95% confidence intervals for the regression estimates.Reviewer #2: This is a well-written manuscript that should be of interest to some of the journal's readers. I have just a few minor comments:1. Page 4, Lines 79-80: "Thus, we hypothesized that VSB . . . " This sentence needs to be re-worded.2. Table 2: add units for PTA and MCL 3. Page 9, Line 170: "The questionnaire results demonstrated statistically significant improvements . . . " Add the word "statistically"4. Page 9, Lines 173-174: "Based on the 20% criterion, 7 patients . . . " add "20% criterion"5. Page 10, Line 194: "only increased MCL was significantly . . . " I think the authors mean "decreased MCL"6. Page 11, Lines 213-214: "There are no known pathophysiological accounts pertaining to . . ." This sentence should be re-worded because I'm not sure what "pathophysiological accounts" means.7. Page 12, Lines 249-251: The last 2 sentences should be deleted because the point of this VAS was NOT to compare current conditions with the patient's memory from 1 year ago. The goal of this VAS was to assess the patient's condition at the time they answered the question: once pre-surgery, and once post-surgery.8. Page 13, Line 261: "relief from hearing gain" Do the authors mean "relief from hearing loss"?**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  18 Dec 2019Reviewer #1: 1. Page 2, lines 24-29: Instead of showing p-values, I suggest to depict point estimates and their 95% confidence intervals.- Thank you for your valuable suggestion. We have reanalyzed the values and modified the paragraph as recommended. - (lines 24-31) “VAS scores for loudness , awareness , and annoyance  showed significant improvements from baseline to 12 months after surgery. In addition, THI scores showed a significant decrease . The average hearing threshold level, WRS, and most comfortable level (MCL) also showed significant improvements at 12 months after surgery . Among the aforementioned factors, changes in MCL were best correlated with those in THI scores .”2. Page 5, lines 91-93: I was curious how many subjects were not included because of the lack of follow-up, audiological tests, and/or self-assessment questionnaires. Inclusion criteria #1-#3 and exclusion criteria #1-#3 would provide the number of study subjects within a 7-month period.- Thank you for your question. We retrospectively reviewed data for patients who received VSB between January 2016 and July 2019. At the first medical examination, we routinely asked the patients about the presence and duration of tinnitus. Although we did not observe the patients for 2 years, the durations (>2 years) were self-reported by the patients. We agree than the current information can cause confusion and have made the relevant changes in the revised manuscript. - (lines 92-93) “…3) stable chronic tinnitus that did not respond to any previous treatments,…”- (lines 98-101) “In total, 47 patients underwent VSB implantation between January 2016 and July 2019. From these, 3 patients were excluded because they had mixed hearing loss. In addition, 16 patients who did not experience tinnitus, 4 who were followed up for less than 1 year, 7 who did not complete the questionnaires, and 1 foreigner for whom a speech discrimination test was not available were excluded.” 3. Table 2:(3a) Column "A-B": I assumed the test for A-B comparison was a paired t test. For paired t tests, I would suggest to replace the percent change with a standard deviation of the difference. The percent change can be easily calculated by readers if necessary.(3b) Column "Probability": I suggest to replace "probability" with 'p-value'.- Thank you for the constructive advice. We have amended the table as per your recommendations. 4. Page 10, lines 189-198: With the sample size of 16, the regression analyses were likely to be under power, meaning that the only significant finding might not be reproducible  or falsely observed . Please discuss this as a limitation in the Discussion section.- Thank you for your valuable comments. We agree that the sample size was too small to perform regression analysis and may have resulted in type II errors. We have mentioned this in the Discussion section and added it as a limitation. - (lines 255-258) “Finally, a sample size compromises the power for the detection of treatment effects and increases the possibility of false-negative results. Thus, the small sample size in this study may be a reason behind some of the insignificant results.” - (lines 272-274) “Furthermore, the sample size was small and may have resulted in type II errors, as discussed earlier. Future studies should also include larger samples to overcome this limitation.”5. Table 3: I suggest to replace the standard errors with 95% confidence intervals for the regression estimates.- Thank you for the constructive advice. We have reanalyzed the values and replaced the standard errors with 95% confidence intervals in Table 3. Reviewer #2: This is a well-written manuscript that should be of interest to some of the journal's readers. I have just a few minor comments: 1. Page 4, Lines 79-80: "Thus, we hypothesized that VSB . . . " This sentence needs to be re-worded.- Thank you for the positive feedback and comments. We have rephrased the sentence in the revised manuscript. - (lines 81-82) “Accordingly, we hypothesized that the use of VSB as a hearing device would have a beneficial effect on tinnitus in patients with sensorineural hearing loss.”2. Table 2: add units for PTA and MCL - Thank you for pointing this out. We have added units for PTA, WRS, and MCL in Table 2. 3. Page 9, Line 170: "The questionnaire results demonstrated statistically significant improvements . . ." Add the word "statistically"4. Page 9, Lines 173-174: "Based on the 20% criterion, 7 patients . . . " add "20% criterion"- Thank you for the valuable suggestions. We have modified the sentences as recommended. - (lines 173-174) “The questionnaire results demonstrated statistically significant improvements after VSB implantation.”- (lines 176-178) “Based on the 20% criterion, 7 patients (43.8%) reported improvement in tinnitus loudness, 8 patients (50.0%) reported no change, and 1 patient (6.2%) reported louder tinnitus after implantation.” 5. Page 10, Line 194: "only increased MCL was significantly . . ." I think the authors mean "decreased MCL"- We apologize for the overlook. We have rectified the error in the revised manuscript. - (lines 196-198) “When we performed multiple regression analysis, only decreased MCL was significantly and positively related to the improvement of THI.”6. Page 11, Lines 213-214: "There are no known pathophysiological accounts pertaining to . . ." This sentence should be re-worded because I'm not sure what "pathophysiological accounts" means.- We apologize for the lack of clarity. We have rephrased the sentence and merged it with the subsequent sentence in the revised manuscript. - (lines 219-221) “The mechanism underlying the beneficial effect of VSB on tinnitus remains unclear, although it may be similar to that of other hearing devices.” 7. Page 12, Lines 249-251: The last 2 sentences should be deleted because the point of this VAS was NOT to compare current conditions with the patient's memory from 1 year ago. The goal of this VAS was to assess the patient's condition at the time they answered the question: once pre-surgery, and once post-surgery.- Thank you for the suggestion. We have deleted the sentences from the revised manuscript. 8. Page 13, Line 261: "relief from hearing gain" Do the authors mean "relief from hearing loss"?- We apologize for the lack of clarity. Because the pathophysiology of tinnitus includes psychological aspects, reducing emotional stress is crucial for managing the condition tinnitus. Emotional stress caused by hearing loss also affects the severity of tinnitus, and hearing gain by means of hearing devices can help in overcoming this emotional stress. Accordingly, we used the expression “relief from hearing gain”, although we agree that the phrase is not clear. We have now modified the sentence in the revised manuscript. - (lines 264-266) “Although tinnitus retraining therapy or direct counseling for tinnitus were not performed in the present study, a frequent fitting schedule with counseling and stress reduction as a consequence of hearing gain would qualify as combined management.”AttachmentResponse to Reviewers.docxSubmitted filename: Click here for additional data file. 17 Jan 2020Effects of Vibrant Soundbridge on tinnitus accompanied by sensorineural hearing lossPONE-D-19-23345R1Dear Dr. Moon,We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.Also, please note that one of the reviewer suggest a very minor revision which can be done during the production stage. Within one week, you will receive an e-mail containing information on the amendments required prior to publication. 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In lines 81-82, I suggest to replace ``beneficial effect'' with `positive association'. Because this was an observational study, I suggest to avoid causal terminologies throughout the manuscript.Reviewer #2: (No Response)**********what does this mean?). If published, this will include your full peer review and any attached files.7. PLOS authors have the option to publish the peer review history of their article (If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.Reviewer #1: NoReviewer #2: No 22 Jan 2020PONE-D-19-23345R1 Effects of Vibrant Soundbridge on tinnitus accompanied by sensorineural hearing loss Dear Dr. Moon:I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! 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Cochlear implants bring sound to people living with permanent hearing loss. But making them accessible to all in need is a major challenge. Andrey Shukshin reports. Stanislav Rubinstein lost his hearing on New Year’s Eve, 2009. The then 20-year-old power company worker had been in an accident and had received treatment for severe burns and spinal damage. But it was not the accident that affected his hearing, it was the antibiotics he was given in hospital.“It happened very quickly,” says Stanislav, who now lives just outside Moscow in the Russian Federation. “December 31, 2009 was the last day I heard anything with my ears.”Five months and 18 operations later, including multiple skin grafts, Stanislav left the hospital able to walk, but cut off from the world of sounds, including the sound of his beloved guitar. He was also cut off from the profession he enjoyed. “I was told I would have to apply for a job as a garment maker,” he says.Three years later he learned of a device known as a cochlear implant.Cochlear implants are hearing devices comprised of an external microphone and speech processor worn just behind the ear that converts sound into electrical stimuli, which are captured electromagnetically by a surgically implanted antenna. The antenna directs the signal to the internal electrodes, which in turn stimulate the auditory nerve.“They are truly ingenious devices,” says Dr Shelly Chadha, who heads the World Health Organization’s (WHO) work on prevention of deafness and hearing loss. “The direct stimulation of the auditory nerve allows the device to overcome hearing loss that occurs when there is damage to the cochlea. This is the most common type of permanent hearing loss and it cannot usually be medically or surgically corrected. So for people with this particular problem, cochlear implants are the only hope of hearing.”Unfortunately, not everyone who could benefit from a cochlear implant gets one. As Chadha explains, there are several reasons for this, the most obvious being their cost. A cochlear implant, including the cost of implant surgery, can be as high as US$ 50 000 in high-income countries, while the external components (transmitter and speech processor) can cost around US$ 9000. No generic version of cochlear implants exists, and the four main manufacturers have kept their prices high even in low- and middle-income countries. In the Russian Federation, for example, a cochlear implant costs up to US$ 18 000, equivalent to roughly two years of the national average income.“It is possible that costs will come down as new producers come into the market,” says Chadha, noting that domestic production in China supplies government programmes there while the federal government of India is discussing the possibility of manufacturing cochlear implants to support the National Programme for Prevention and Control of Deafness.“Post-operative rehabilitation may take five to seven years and sometimes a lifetime.”Victoria MukhinaBut even if the price of cochlear implants does come down, it is unlikely to have a major impact on the overall cost of implementing effective cochlear implant programmes and thus the availability of devices for people who need them. For example, the Russian government started supporting cochlear implantation with the implantation of two devices in 1987 increasing to 100 implantations in 2005. Despite a sharp economic downturn since 2008, the government has continued to support implantation, funding around 1000 cochlear implants a year out of the federal budget. In the absence of official statistics, it is difficult to establish how close this support comes to meeting the demand. However, according to Dr George Tavartkiladze, founder and director of the National Research Centre for Audiology and Hearing Rehabilitation, demand currently stands at around 3000-35 000 implants annually.Roughly 90% of the implants are reserved for children. Maternity hospitals with a capacity of more than 1000 births per year, regional audiological centres and pediatric outpatient clinics are equipped for newborn hearing screening. Tavartkiladze estimates that initial screening coverage is close to universal (98%) in the majority of the federation’s 85 administrative regions (krai). He points out however, that coverage of second stage screening, which involves a full diagnostic test, is done only in the regional audiological centres, and currently stands at around 80%.Why this emphasis on treating children? Tavartkiladze explains that children are considered more likely to benefit from the procedure than adults who have been deaf from birth. However, adults who have lost their hearing due to illnesses such as meningitis or to head injury, are eligible to have an implant fitted within a few months.According to Tavartkiladze, almost all implantations are done at six federal institutions, three of which are located in Moscow, two in Saint Petersburg, and only one, the Federal Siberian Research and Clinical Center, in Krasnoyarsk. “The operation itself has become a routine procedure,” he says, underlining the high success rate achieved by surgeons and their teams.While the Russian Federation’s cochlear implant programme is clearly laudable in many ways, it also has some important limitations. To begin with, in most cases, the government only funds one implant per person . This limits the recipient’s ability to participate in everyday life. “The benefit of using implants on both ears, so-called binaural implantation, is in gaining the ability to localize sound and in significantly higher speech intelligibility in acoustically difficult environments, such as noisy rooms,” says Victoria Mukhina, head of Tosha&Co, a private rehabilitation centre for children with cochlear implants, which is located in Fryazino just outside Moscow.Mukhina believes it is vital that the government support binaural implantation. “It’s a question of resources,” she says. “We must get together and get the government to fund two implants on a massive scale.” She also believes that it is essential that the government expand implantations for adults, pointing out that in most developed countries adults account for more than half of the implantations undertaken.The fact that the federal government tends to fund only the first post-operative rehabilitation session is also a matter of concern. The Moscow region benefits from the Federal Centre for Rehabilitation of Children with Hearing Loss, where all children with cochlear implants can get two weeks of rehabilitation sessions free of charge, but outside the Moscow region no such service exists.This is a problem, as Mukhina explains. “The implant operation itself only addresses part of the patient’s needs,” she says, noting that when the implant is switched on, the external speech processor does not result in the sudden restoration of hearing. The sounds produced by the speech processor have to be reinterpreted. “Cutting-edge technologies are great, but they need to be supported by a properly trained and motivated workforce.”Alexander Ivanov“This process is easier for the adults and children who lose their hearing after they have already mastered speech. For those who become deaf before they have learned how to speak or who are born with hearing loss, the earlier they get the implant, the better the results they can achieve.” Mukhina says. “Post-operative rehabilitation may take five to seven years and sometimes a lifetime,” she adds.There are currently five main medical institutions undertaking medical rehabilitation, and once again, services are highly centralized, with four of the centres located in Moscow, and one in Saint Petersburg. Sufficient staff are also needed. Learning how to hear and speak can require the support of audio-therapists, psychologists and neurologists. “Cutting-edge technologies are great, but they need to be supported by a properly trained and motivated workforce,” says Alexander Ivanov, head of rehabilitation at the Russian Deaf Association. “To focus on just one speciality, we currently have a 30% shortage of audiologists across the country.” “I have a feeling that officials thought cochlear implants were this wonderful, quick-fix, high-impact solution,” says Ivanov, “but that is not how things work.”Stanislav Rubinstein would agree, in part. It took him seven years to get back to full participation in everyday life; but he would consider “wonderful” and “high-impact” entirely appropriate descriptions of the devices he now proudly wears.Today, apart from those devices there is no indication of his deafness. After receiving a job offer from the local distributor of a major international cochlear implant producer, he moved to a town outside Moscow, where he is now working as a technical assistant.“I am so happy with my implants that if somebody told me there is a wonder surgeon who could try and get my natural sense of hearing back, I wouldn’t go for that. I lead a normal life, I communicate freely, I have a great job, I enjoy music just like before and I can play my beloved guitar like I used to,” he says.