Severe birth asphyxia (apgar < 7 at the 5th minute of birth) is recognized as a hearing loss risk factor by the joint committee on infant hearing (JCIH). About half of the newborns with hearing loss do not indicate any sign and risk factor at birth. Accordingly, the joint committee recommended performance of hearing screening test in 2000, especially for babies born with risk factors.The aim of this study was to evaluate hearing loss in asphyxiated neonates. Early diagnosis would result in early treatment of these newborns.We assessed the relationship between asphyxia and hearing impairment in newborns admitted to a referral hospital, Tehran, Iran within 3 years (2003 - 2006). Hearing problems were diagnosed and followed by otoacoustic emission (OAE) in the third and fifth days of birth. Asphyxiated neonates with abnormal OAE were referred to an ENT specialist; second OAE and tympanometry were carried out after 2 weeks. Based on the results, newborns underwent treatment or were discharged.Of 149 asphyxiated neonates, 80 had mean first minute apgar score of 4.01, and mean 5th minute score was 7.24. Two percent (3/149) of asphyxiated neonates had abnormal OAEs. No statistical correlation was found between the 5th minute apgar score and abnormal OAE . However, a significant relationship between the mean birth weight and abnormal OAE  was found.It seems that birth asphyxia is not correlated with hearing loss. Hearing loss is a common anomaly presenting at birth (1/500 - 1/1000) . Based oChildren with impaired hearing, present delays in language learning and general development . This prThe aim of this study was to evaluate hearing loss in asphyxiated neonates. Early diagnosis would result in early treatment of these newborns.Newborn hearing screening by OAE was initiated in Imam Khomeini hospital , Tehran University of Medical Sciences (TUMS), as a neonatal referral and academic center,  (2003 - 2006). The target population included every asphyxiated newborn with first minute Apgar score < 5, and/or 5th minute Apgar score < 6. This case-series study was approved as a student thesis by the research and medical ethics committee of TUMS to the Helsinki declaration. Some variables of mothers and babies were collected in questionnaires .OAE screening was performed by Echo Screen TE machine  that uses transient-evoked otoacoustic emission. At the end of the test, the result was shown on the screen as “PASS” when there was OAE response (response to the stimulus at > 35 dBHL (decibels Hearing Level)), and REFER when there was no response to the stimulus . ScreeniIn this study 149 newborns with birth asphyxia were evaluated for hearing loss. Demographic and clinical characteristics of the asphyxiated neonates are shown in The highest and lowest gestational ages were reported 40 and 26 weeks, respectively. Newborns with abnormal tests represented mean gestational age of 34.12 ± 3.7 weeks and mean birth weight of 2014.21 ± 344 g compared with those with normal OAEs with mean gestational age of 36.05 ± 3.7 weeks and mean birth weight of 2551.35 ± 446 g . Of 149 cases, 80 had first minute Apgar score of less than 5, 28 of which (35%) failed the test (mean Apgar score of first minute was 4.01), but among these neonates only 24 had 5th minute Apgar score of less than 6 (mean Apgar score = 7.24), and 5 (22.7%) failed the test. But in 69 cases, first minute and 5th minute Apgar scores were higher than 5 and 6 respectively, asphyxia diagnosis based on clinical signs of the first hours of birth, fetal bradycardia, and meconium stained amniotic fluid. It is noteworthy that no significant relationship was found between abnormal OAE and 5th minute Apgar score of less than 6 .Hearing loss can be considered as one of the most important birth defects. Birth asphyxia and ischemia have often been thought to be major causes of early hearing loss or deafness. Experiments have confirmed/shown that hypoxia induces the ABR elevation threshold in rat and cat neonates . SeveralOf 149 neonates in our study, approximately 2% (3/149) had abnormal OAEs, but we may underestimate this rate, since some of our cases with abnormal OAEs did not continue the follow up. Our finding was more than the incidence of hearing loss at birth (1/500 - 1/1000), however, in another study the rate of abnormal OAE at high-risk newborns was reported approximately 2% . FurtherBabies who were born by C/S had more abnormal OAE in comparison with NVD-born babies . It seems that spinal or epidural anesthesia had some effects on the hearing system . In addiWe also found that there is no significant correlation between congenital hearing loss and exclusively the 5th mean Apgar score as an important feature for severity of asphyxia. This finding is compatible with former studies, too . MoreoveAs a matter of fact, evaluation of the exact etiology of neonatal hearing loss in children with complicated deliveries is difficult because of the large number of causative factors involved. However, it seems that birth asphyxia is not correlated with hearing loss; at the same time it is impossible to say that the relative importance of different factors and their interactions are clearly identified. Indeed, given the results of newborns hearing loss screening in majority of countries, we are no longer in doubt that hearing screening should be performed for all newborns, especially the ones at risk during neonatal period.


Noise-induced hearing loss (NIHL) is a major concern in the non-manufacturing industries. This study aimed to investigate the occupational noise exposure and the NIHL among Chinese restaurant workers and entertainment employees working in the service industry in Hong Kong.This cross-sectional survey involved a total of 1,670 participants. Among them, 937 were randomly selected from the workers of Chinese restaurants and 733 were selected from workers in three entertainment sectors: radio and television stations; cultural performance halls or auditoria of the Leisure and Cultural Services Department (LCSD); and karaoke bars. Noise exposure levels were measured in the sampled restaurants and entertainment sectors. Each participant received an audiometric screening test. Those who were found to have abnormalities were required to take another diagnostic test in the health center. The “Klockhoff digit” method was used to classify NIHL in the present study.The main source of noise inside restaurants was the stoves. The mean hearing thresholds showed a typical dip at 3 to 6 KHz and a substantial proportion (23.7%) of the workers fulfilled the criteria for presumptive NIHL. For entertainment sectors, employees in radio and television stations generally had higher exposure levels than those in the halls or auditoria of the LCSD and karaoke bars. The mean hearing thresholds showed a typical dip at 6 KHz and a substantial proportion of the employees fulfilled the criteria for presumptive NIHL . Being male, older, and having longer service and daily alcohol consumption were associated with noise-induced hearing impairment both in restaurant workers and entertainment employees.Excessive noise exposure is common in the Chinese restaurant and entertainment industries and a substantial proportion of restaurant workers and entertainment employees suffer from NIHL. Comprehensive hearing conservation programs should be introduced to the service industry in Hong Kong. Occupational NIHL has been regarded as a global public health problem, and systematic research on occupational NIHL was performed as early as the late 19th century However, few studies have been carried out to ascertain the size of the exposed group and the magnitude of the problem in the service sector. Employees who work in Chinese restaurants in Hong Kong are an obvious target group, because noise-generating pressurized gas stoves are frequently used in Chinese restaurants for Chinese-style cooking. At present, little data on noise in Chinese restaurants are available. A systematic and scientific evaluation of noise levels in Chinese restaurants and the collection of baseline prevalence data on the hearing levels of the workers are essential for developing occupational health control measures.In addition to restaurant workers, many studies have shown that there is a risk of hearing loss among musicians and teenagers who listen to loud music The Hong Kong Standard Industrial Classifications code for Chinese restaurants is 641 Service workers: workers in the service areas, including managers, captains, waiters/waitresses, pantry helpers, dim sum sales staff, barbecue counter staff, fish tank caretakers, dish-up, clerks, and accountantsCooks: including chief cooks, second cooks, dim sum cooks, and barbecue cooksDishwashersKitchen workers: workers inside the kitchens other than cooks.Based on an estimated 30% prevalence of noise-induced hearing impairment among the exposed workers Noise assessment included measuring the noise levels in selected working environments, and measuring the cumulative personal noise exposures for some participants with job titles including cook and dishwasher, which are a typical position having a high risk of noise exposure in Chinese restaurants.Environmental noise levels were measured by using precision sound level meters  in various locations where the workers were working. The measurements were then integrated to give an A-weighted sound pressure level (LAeq) for the time period of the measurements.d values). The computing capability of the dosimeter allowed a complete real-time record of the sound levels which the workers were exposed to during the measurement period to be statistically analyzed. The personal exposure data were then transferred from the dosimeters to a personal notebook computer  and they were then processed by using the software supplied with the dosimeters.Regarding cumulative personal noise exposure, “B & K” noise dosimeters  were used to measure the participants' personal daily noise exposure levels  radio and television stations; (2) cultural performance halls or auditoria of the LCSD; and (3) karaoke bars. One radio and television station, 11 halls or auditoria of the LCSD and two karaoke bars were selected, and their employees were invited to participate in this study. The response rate in each sector varied. For the radio and television stations, a total of 428 employees were invited and 262 of them agreed to participate (response rate: 61.2%); for the 11 halls or auditoria of the LCSD, 613 employees were invited and 438 of them agreed to participate (response rate: 71.5%); for the two karaoke bars, 100 employees were invited and 33 of them agreed to participate (response rate: 33.0%). A total of 733 participants were therefore included in the analysis. The study protocol was reviewed and approved by the Survey and Behavioural Research Ethics Committee of Chinese University of Hong Kong. Signed informed consent was obtained from the participant beforehand.Noise assessment referred to the measurements of noise levels for (1) typical radio program presenters who wore headsets during work, and (2) typical participants who did not wear headsets during work.A special method was used to assess the personal noise exposures for radio program presenters who wore headsets while carrying out their duties. The sound pressure levels were measured by using a precision sound level meter  via an artificial ear (CUOI) with the earphones  plugged into the monitor interphase that was parallel in output and subjected to adjustment with the headset worn by the presenters. In other words, we measured the sound levels in the artificial ear, which had the same levels of sound pressure as in the ears of the person who wore the headset.For participants who did not wear headsets, cumulative personal noise exposures were assessed and methods. The methods and equipments were the same as those used for restaurant cooks and dishwashers, described above.The audiometric tests consisted of a screening test in the field and a diagnostic test in the hearing test center. The screening test was performed for all participants . Those who were found to have abnormalities during the screening test were then requested to attend the diagnostic audiometric test.A standard questionnaire was used for all participants during the audiometric tests to collect information such as personal particulars, current and past occupational noise exposures, noise exposures other than from their occupation and the use of ear protectors. An otoscopic examination of the external ear canals and eardrums was then performed and any abnormalities were noted.Audiometric screening examinations were carried out in the field. For all participants, pure tone air conduction hearing thresholds at different frequencies were measured manually by the abridged ascending method following a standard procedure in accordance with BS 6655: 1986. The hearing thresholds were measured for both ears at the following octave frequencies: 500, 1,000, 2,000, 3,000, 4,000, 6,000, and 8,000 Hz. A portable audiometer  with earphones  was used to perform the audiometric screening test. The earphones were equipped with audiocups  to enable testing in quiet rooms at workplaces. The rooms used for the audiometric testing were either VIP rooms or the managers' rooms. The background sound pressure levels were monitored by using a precision sound level meter  with a type NX-05 octave band filter unit. Sound levels (LAeq) in these testing rooms varied considerably, ranging from 46–74 dBA. For low frequencies (∼500 Hz), the noise levels of most locations ranged from 44–75 dBA, while at higher frequencies (∼8 KHz) the levels ranged from 16–63 dBA.Participants who were found to have abnormalities (having a hearing threshold >25 dBA at any frequency in either ear) during the screening test were then requested to attend an audiometric diagnostic test. The diagnostic test included the pure tone air conduction hearing thresholds at the following octave frequencies 500, 1,000, 2,000, 3,000, 4,000, 6,000, and 8,000 Hz for both ears. Bone conduction audiometry was performed by using the method described by the British Society of Audiology The audiometric diagnostic test was carried out inside a sound-proof audiometric booth  by using a Madsen audiometer  at the Department of Community and Family Medicine, Lek Yuen Health Centre in Shatin, or at the Hong Kong Federation of Trade Unions Clinic  in Mongkok.Objective calibrations of all audiometers were carried out once every six months to adjust the sound pressure output in the left and right earphones by using an artificial ear. Biological calibration checks of audiometer functions were carried out before the start of the tests on each day of fieldwork.et al.The classification of hearing defects used in this study was suggested by Klockhoff The term ‘Presumptive Noise Induced Hearing Loss’ (P-NIHL) was used when high tone hearing impairment  was detected in any subject in this study. There are three levels of P-NIHL, namely, slight, moderate, and severe high-tone loss. According to the above coding classification, P-NIHL could be made up of combinations with “12”, “21”, “13”, “31”, “14”, “41”, “22”, “23”, “32”, “24”, “42”, “33”, “34”, “43”, and “44” when the differences in the average hearing levels at 3,000, 4,000, and 6,000 Hz between the better and poorer ear did not exceed 30 dBA To indicate the prevalence of moderate and severe high tone loss in our study population, we used the term “High Tone Loss” (HTL) in this report, which combined P-NIHL coding “33”, “34”, “43”, and “44”.The double entry of data into computers was done independently by two research assistants and compared subsequently. Any mismatch was corrected after checking the corresponding questionnaire and the test record. Descriptive statistics were used for calculating the means of the continuous variables and the prevalence of the categorical variables. The logistic model was used for calculating the odds ratios. We considered p values of <0.05 to be statistically significant, and 95% confidence intervals were also presented. All data analyses were performed by using the Statistical Package for the Social Science .The mean age (SD) of the 733 entertainment employees was 38.6 (9.4) years, which was younger than that of the 937 restaurant workers (their mean age was 41.9 (11.7) years). There were more females working in the Chinese restaurants. The mean duration (SD) of working in the entertainment sector was 7.6 (7.1) years, while the service duration for restaurant workers was 10.3 (9.5) years. The general characteristics of the entertainment employees and restaurant workers are presented in The main source of noise inside restaurants was from the stoves. Other sources included dishwashing machines and ventilation systems, such as fume extractors. The measurements of environmental noise in different areas are shown in A total of 22 personal dosimetric measurements were taken from 20 cooks and two dishwashers . Higher A total of 34 personal dosimetric measurements of typical entertainment employees who did not wear headsets were taken from various job groups. Noise levels ranged from 74.3 to 101.9 dBA. Filming with headphone (TV) and Carpenter (TV) had the highest exposure to mean noise levels. The results of dosimetric measurements for the entertainment employees are also presented in The noise levels of the radio program presenters who wore headsets are presented in The mean hearing thresholds by frequency for restaurant workers stratified by gender are shown in Panel A in The prevalence of P-NIHL and HTL, were 23.7% (95% CI: 21.0–26.4) and 11.3% (95% CI: 9.3–13.3) among restaurant workers, respectively. For entertainment employees, the corresponding figures were 38.6% (95% CI: 35.1–42.1%) and 7.0% (95% CI: 5.2–8.8%). Our survey examined noise exposures and hearing impairment in Chinese restaurant workers and entertainment employees in Hong Kong. Our results show that noise exposure is common and excessive in Chinese restaurants and in the entertainment industry in Hong Kong. From our representative sample of Chinese restaurant workers we estimated that about 47% of workers in Chinese restaurants are exposed to noise levels of above 85 dB(A). The mean hearing thresholds show a typical dip at 3 to 6 KHz and a substantial proportion of restaurant workers fulfilled the criteria for P-NIHL and HTL (23.7% suffer from P-NIHK and 11.3% suffer from HTL) Regarding the noise exposures and prevalence of hearing loss in the manufacturing industry in Hong Kong, previous studies have shown that 38% of workers were exposed to noise levels of 90 dB(A) or more, and that 19% suffered from HTL, which was higher than the HTL prevalence (7.0%) in our entertainment employees and the HTL prevalence (11.3%) in Chinese restaurant workers, but there are no data on P-NIHL in this study et al. in San Francisco showed that the noise levels generally ranged from 60–80 dB(A), with the peak at 87 dB(A) There is a little information about NIHL for restaurants in other countries. The study conducted by Lebo et al. reported that environmental noises and headphone styles were associated with increased preferred listening levels among the adults using MP3 players Hitherto, there has been limited information on hearing loss on employees in the entertainment industries worldwide. Some overseas studies for employees working at music venues showed that the prevalence generally ranged from 29–50%. However, the sample sizes of these studies were small with there being 14–82 participants in each study Aging has been well documented as an important risk factor for hearing loss. However, presbycusis usually shows a pattern on an audiogram different from that of NIHL. Generally presbycusis shows the greatest threshold shifts at higher frequencies, leading to a downward sloping curve without a “notch” et al. reported an association between hearing levels and smoking Our results show that being male, and older were significantly associated with an increased risk of hearing impairment in both restaurant workers and entertainment employees. These findings are in conformity with previous studies There are several disadvantages in our study which limited us for thoroughly assessing the relationship of NIHL with noise exposures: (1) due to the shortage of resources, information on many potential risk factors could not be collected, and we explored only the associations between NIHL and some demographic factors as well as smoking and drinking. (2) the cross-sectional nature did not allow us to assess any temporal relationship of NIHL with noise level as well as the potential risk factors. Prospective cohort studies are required to thoroughly assess the relationship between sound exposures and NIHL, as well as the effects of other risk factors/confounders on the relationship; and (3) the current measurements of noise exposure levels might not accurately reflect past exposures. Other limitations in our survey include the low response rate for karaoke bars, the non-representative sampling in the entertainment industry, and small sample size of each sub-sector in the entertainment industry. However, because of the wide scope and various job natures in the entertainment industry, a representative sample is difficult to obtain unless a census can be carried out for the whole entertainment industry.Our results have important public health implications. NIHL is permanent and irreversible, but actually it is completely preventable


Background. Electric-acoustic stimulation (EAS) is an excellent choice for people with residual hearing in low frequencies but not high frequencies and who derive insufficient benefit from hearing aids. For EAS to be effective, subjects' residual hearing must be preserved during cochlear implant (CI) surgery. Methods. We implanted 6 subjects with a CI. We used a special surgical technique and an electrode designed to be atraumatic. Subjects' rates of residual hearing preservation were measured 3 times postoperatively, lastly after at least a year of implant experience. Subjects' aided speech perception was tested pre- and postoperatively with a sentence test in quiet. Subjects' subjective responses assessed after a year of EAS or CI experience. Results. 4 subjects had total or partial residual hearing preservation; 2 subjects had total residual hearing loss. All subjects' hearing and speech perception benefited from cochlear implantation. CI diminished or eliminated tinnitus in all 4 subjects who had it preoperatively. 5 subjects reported great satisfaction with their new device. Conclusions. When we have more experience with our surgical technique we are confident we will be able to report increased rates of residual hearing preservation. Hopefully, our study will raise the profile of EAS in Brazil and Latin/South America. Just over a decade ago people with sensorineural hearing loss had 2 main hearing (re)habilitation options: (1) a hearing aid (HA) if they had mild to moderate hearing loss and (2) a cochlear implant (CI) if they had severe to profound hearing loss. These 2 device options improved most users' hearing. However, people who could hear in the low frequencies (up to 1000 Hz) but not the medium and high frequencies—the downward or “ski slope” audiogram—had too much high frequency hearing loss to benefit from their hearing aid(s) but were not CI candidates because surgeons feared the surgery would destroy their residual hearing.A solution for such people is electric-acoustic stimulation (EAS), a concept developed by von Ilberg and colleagues in 1999 –4 and be“Soft” surgery was first described by Lehnhardt and Laszig in 1994  and mult24 (formerly known as the FLEXEAS), as it is the electrode we used in our study, recently surgeons have used it to achieve partial or complete hearing preservation in 100% of their study subjects [Electrode design  is also critical to reducing cochlear trauma –21; bothsubjects . Althougsubjects , it was 24 electrodes. Study inclusion criteria were as follows: all subjects had to (1) be older than 18 years, (2) have sensorineural bilateral hearing loss with little or no benefit from HA (less than 40% of auditory discrimination in monosyllables), (3) have pure-tone thresholds of ≤60 dB hearing loss in at least 1 frequency between 250 and 500 Hz and of ≥80 dB in frequencies above 1000 Hz, (4) have had stable hearing loss for at least the past two years, and lastly (5) pass a psychological examination ensuring they had realistic expectations about the potential benefits of receiving a cochlear implant and/or using EAS (6 subjects (mean age 47 years) were implanted with MED-EL cochlear implants with FLEXsing EAS . All subWe used the same surgical technique on all subjects. The technique, which we have named the UNICAMP approach, is a mastoidectomy approach. It is based on techniques developed in various otology centers. Patients were under general anesthesia, tracheal intubation, and placed in a supine position with their head turned to the contralateral side. The operative field was prepared through extensive shaving, cleaned with chlorhexidine 2%, and the attachment of the electrodes to monitor CN VII.The main landmarks are marked: tip of the mastoid, temporal line, retroauricular incision line, area of the internal component, and area of the microphone with the help of an implant template;antisepsis with 0.2% aqueous chlorhexidine, placement of sterile drapes and steri-drape 2; rectilinear retroauricular incision and dissection along anatomical planes; preparation of a “cross” Palva flap  raising the four segments of the flap over the subperiosteal plane; removal of small fragments of fascia and temporal muscle to occlude the cochleostomy;simple mastoidectomy, identifying the lateral semicircular canal, the short ramus of the anvil, the posterior wall of the outer ear canal, the tegmen timpani, and the lateral sinus; gathering a small amount of bone dust;thinning of the posterior wall of the outer ear canal, posterior tympanotomy, preservation of the incus buttress; preparation of the receiver bed for the implant on the squamous portion of the temporal bone (well) using a specific implant template;irrigation of the cavity with povidone-iodine (10% povidone-iodine/1% active iodine) for two minutes followed by abundant irrigation with lactated Ringer's solution;irrigation of the cavity with ciprofloxacin (4 mg/mL) for two minutes followed by irrigation with lactated Ringer's solution;intravenous administration of dexamethasone (8 mg) before approaching the inner ear via a cochleostomy or through the round window;application of topical triamcinolone (40 mg/mL) over the round window; opening the membrane of the round window; if this approach is impossible, the endosteum is opened by means of a cochleostomy;positioning of the implant into the prior drilled bed;Preparation of the fascia graft; making a pinhole central orifice to allow the electrode to pass snugly to be placed in the cochleostomy/round window site;insertion of the electrode slowly and continuously during three minutes;positioning the muscle graft around the electrode to seal the cochleostomy; placing bone dust to close the posterior tympanotomy;positioning the ground electrode under the muscle-periosteum flap;closure with Vicryl 3.0 sutures on the Palva flap planes and subcutaneous tissue; skin closure with Nylon 4.0;cleaning of the patient and placing an external compressive dressing;impedance testing, neural response telemetry (NRT), and a transorbital incidence radiograph are done to confirm the position of the intracochlear electrode.We used a micropore to isolate the operative field from the rest of the scalp and gave prophylaxis with cefazolin (50 mg/kg) intravenously during induction of anesthesia.The same surgeon and surgical team performed all six surgeries.All subjects had unaided pure-tone audiometry tests at 250, 500, 1000, 2000, 3000, 4000, 6000, and 8000 Hz  at activation, (2) 6.5–7 months after activation, and (3) approximately 7 months after their previous test. We defined “residual hearing preservation” in three ways:Lastly, to measure efficacy, all subjects had free field warble tone tests with EAS or CI-only  at the same postoperative intervals as were their unaided tests.Preoperatively, all subjects took a speech perception test the same day as their implantation. We used a speech perception sentence test based on one developed by Bevilacqua et al. from several English language tests . SubjectPostoperatively, all subjects repeated the speech perception test after at least 1 year of CI experience. Tests were done in subject's best-aided condition: EAS or CI-only, depending on their postoperative residual hearing. The same audiologist conducted all the pre- and postoperative tests.When the subjects did their postoperative speech tests they were asked to rate the quality of their experience with EAS/a CI over the past year on a Likert scale scored 0 to 10. A score of 0 indicated the user regretted the intervention, would not recommend it to others, and felt he/she had been better off in the past with their hearing aids. A score of 10 indicated the user was completely satisfied with the intervention and would strongly recommend it.The institutional review board approved this study and all subjects gave written informed consent.EAS electrode. At no frequency or test interval did any subject's aided or unaided PTA score vary by more than 10 dB from their scores at the same frequency in either of the other 2 tests. For the sake of convenience, the graphs show their PTA scores at their last postoperative test  after 14 months CI experience.He rated the quality of his experience a 9 on Likert scale, indicating he was very pleased with the intervention.He had suffered from idiopathic hearing loss for 10 years and had been using hearing aids for 8 months without benefit. Unlike the other 5 subjects, he was implanted via cochleostomy instead of round window because we could not visualize the round window. The subject was fit with a CI only.As you can see from his pre- and postoperative hearing test results, he derived real benefit from implantation. We, however, failed to preserve his residual hearing.His speech test score improved from 0% preoperatively to 16% (with CI-only) after 14 months CI experience. We attribute this relatively poor speaking perception score to the fact that he (1) lost (or had stolen) his external component and so was without CI experience for approximately 11 of the 14.5 months between his activation and post-op speech test and (2) he missed audiological rehabilitation sessions.He rated the quality of his experience a 6 on the Likert scale, indicating he was mildly pleased with the intervention.She had suffered from idiopathic hearing loss for 10 years and had been using hearing aids for the past 6 years, with little benefit. Her preoperative tinnitus and dizziness were eliminated postoperatively. The subject was fit with a CI only.As you can see from her pre- and postoperative hearing test results, she derived real benefit from implantation. We, however, failed to preserve her residual hearing.Her speech test score improved from 28% preoperatively to 78% (with CI-only) after 13 months CI experience.She rated the quality of her experience a 9 on the Likert scale, indicating she was very pleased with the intervention.He had suffered from idiopathic hearing loss for 8 years and had been using hearing aids for 4 years in his right ear and 1 year in his left ear, with little benefit. He had also been treated with corticosteroids, with little improvement. The subject was fit with EAS with the CI cut-off frequency set at 700 Hz. His preoperative tinnitus and dizziness were eliminated postoperatively. As you can see from his pre- and post-op hearing test results, he derived real benefit from implantation. We achieved total residual hearing preservation.His speech test score improved from 8% preoperatively to 84% (with EAS on) after 15 months CI experience.He rated the quality of his experience a 10 on the Likert scale, indicating he was extremely pleased with the intervention.He had suffered idiopathic hearing loss for 15 years and had using hearing aids for 5 years, with little benefit. The subject was fit with EAS with the CI cut-off frequency set to 350 Hz. His preoperative tinnitus was eliminated postoperatively.As you can see from the pre- and postoperative data, he derived real benefit from implantation. We achieved partial residual hearing preservation.His speech test score improved from 0% preoperatively to 82% (with EAS on) after 12 months CI experience.He rated the quality of his experience a 10 on the Likert scale, indicating he was extremely pleased with the intervention.He had suffered from idiopathic hearing loss for 15 years and had been using hearing aids for 5 years, with little benefit. The subject was fit with EAS with the CI cut-off frequency set to 1 kHz.As you can see from the pre- and postoperative hearing test results, he derived some benefit from implantation. We achieved partial residual hearing preservation.His speech test score improved from 10% preoperatively to 40% (with EAS) after 13 months CI experience. Despite his relatively poor postoperative speech perception score, the audiologist reports that he is improving.He rated the quality of his experience an 8 on the Likert scale, indicating he was very pleased with the intervention.Some benefits of the good outcomes are represented by the individual speech tests of all subjects Tables .Of our 6 subjects, 1 had total residual hearing preservation, 3 had partial residual hearing preservation, and 2 had total residual hearing loss: a residual hearing preservation success rate of 4/6. Looked at another way, postoperatively, 4 subjects will benefit from EAS and 2 subjects (2 and 3) are no longer partially deaf and thus no longer EAS candidates, although they enjoy better hearing from their CI than they had had from their HA before implantation. All subjects who actually had 1-year implant experience had greatly improved postoperative speech perception scores. 3 EAS users  scored between 82% and 84%, similar to the results of previous studies . SubjectWe demonstrated that cochlear implant surgery done with the aim of preserving residual hearing is highly beneficial to the hearing lives of the partially deaf—as evidenced in their extremely positive Likert scale responses—even when we fail to preserve their residual hearing. We, nonetheless, fell short of our lofty goal of 100% residual hearing preservation. We attribute this to 2 primary causes. Firstly, living in and working in Brazil, we have had very few EAS cases and less experience with hearing preservation surgery than do the surgeons whose results are featured in other articles. EAS was developed in Germany as recently as 1999 and, correspondingly, most experts come from Central Europe . We (UNICAMP) are the only team in Brazil—a nation of almost 200 million—that does hearing preservation surgeries like this. The surgical technique is difficult and we are confident that with experience, and better-suited cases, we will improve our success rate. Our results should be seen in this context: a regional beginning.Secondly, we had limited access to suitable EAS candidates. All subjects had idiopathic hearing loss and not all of them were “true” EAS candidates. Subjects 1 and 5 had preoperative scores of 70 dB and 80 db, respectively, at 500 Hz, whereas the maximum indication for EAS is 60 dB at 500 Hz. We implanted them anyway—as other surgical teams have done , 26—becaSOFT electrodes and the hearing preservation round window technique they developed and described in 2007 [EAS electrodes [Our residual hearing preservation results were below those of other similar studies. Skarzynski et al.  partiall in 2007 . Arnoldnectrodes . Gstoettectrodes . They usAdditionally, the provision of a CI decreased subjects' tinnitus and dizziness. 4 subjects  suffered from preoperative tinnitus. Postoperatively, only subject 1 still had tinnitus, but he said it was “not too bad.” These results are consistent with past studies –31, whicWe strongly believe that EAS has an important place in the future of otology. While it is entering its teenage years in Central Europe, it is still in its infancy in Brazil. We hope our results will raise the profile of EAS here and hearing preservation surgery and help make it more common. Hopefully, with more experience and sufficient subjects, we—and other new teams—will soon be reporting residual hearing preservation results comparable to those of currently well-established surgeons.


Objective. Polycystic ovarian syndrome (PCOS) is the most commonendocrine disorder affecting 5–10% of women in reproductive age. Insulin resistance,dyslipidemia, glucose intolerance, hypertension, and obesity are metabolic disordersaccompanying the syndrome. PCOS is a chronic proinflammatory state and the diseaseis associated with endothelial dysfunction. In diseases withendothelial damage, hearing in high frequencies are mostly effected in early stages. We evaluated extended high frequency hearing loss in PCOS patients.Material Methods. Forty women diagnosed as PCOS and 25 healthy controls were included in this study. Ageand BMI of PCOS and control groups were comparable. Each subject was tested with low (250–2000 Hz),high (4000–8000 Hz), and extended high frequency audiometry (8000–20000). Hormonal and biochemical values including LH, LH/FSH,testosterone, fasting glucose, fasting insulin, HOMA-I, and CRP were calculated. Results. PCOS patients showed high levels of LH, LH/FSH, testosterone, fasting insulin, glucose, HOMA-I, and CRP levels.The hearing thresholds of the groups were similar at frequencies of 250, 500, 1000, 2000,and 4000 Hz; statistically significant difference was observed in 8000–14000 Hz in PCOS group comparedto control group. Conclusion. PCOS patients have hearing impairment especially in extended high frequencies.Further studies are needed to help elucidate the mechanism behind hearing impairment in association with PCOS. Polycystic ovarian syndrome (PCOS) is the most common endocrine disorder affecting 5–10% of women in reproductive age . PCOS isRecently, a study conducted by Oghan et al. showed that patients with PCOS have sensorineural hearing loss in high frequencies . They poThe study was carried out at Dumlupinar University Kutahya Evliya Celebi Training and Research Hospital gynecology outpatient clinic. Forty patients with the PCOS and twenty-five healthy subjects were enrolled in this prospective study. The control group consisted of patients who admitted to hospital for routine gynecological examination.We included healthy women as controls with normal menstrual cycles, with no evidence of hyperandrogenism, and with normal ovarian morphology on pelvic ultrasonography. Ferriman Gallwey scores of all control patients were under 8. PCOS was defined as the presence of two of the following three features after the exclusion of other etiologies , (i) olihttp://www.wma.net/). Regional Ethical Committee approved the study. All patients gave oral and written informed consent prior to the examination.The study was conducted according to the guidelines for clinical studies described in the Declaration of Helsinki  and diastolic blood pressure (DBP) were measured twice in the right arm in a relaxed sitting position. Two measurements were taken 15 minutes apart. The average of two was used.Body mass index (BMI) was calculated as weight (kg)/height (m)Blood samples were collected during early follicular phase of menstrual cycle after at least 12-hour fasting. Levels of glucose, insulin, and hormone profile (follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2), total and free testosterone , and prolactin (PRL)) were determined. Plasma glucose was determined with the glucose hexokinase method . Hormone profile is measured with electrochemiluminescence assays .Plasma concentrations of insulin were measured by chemi-luminescent immunoassay . Insulin resistance was measured with HOMA-IR (homeostasis model assessment for insulin resistance) .All the participants were subjected to careful ear examination to identify any abnormalities that may interfere with hearing such as a perforated tympanic membrane or other middle ear pathologies. All subjects had normal immittance audiometry results. Each participant was tested with Low Frequency Audiometry (LFA); 125 Hz to 2 kHz, High Frequency Audiometry (HFA); 4 kHz to 8 kHz, and Extended High Frequency Audiometry (EHFA); 9 kHz to 20 kHz. Audiometry was performed by an expert audiologist blinded to the study.t-test. The chosen significance level was P < 0.05.All statistical analyses were performed using the SPSS for Windows, version 17.0. Nonparametric tests were used as the variable hearing threshold had abnormal distribution due to data dispersion and lack of distribution symmetry. After testing the normal distribution, comparisons between the groups were tested using the 2 and 24.4 ± 3.8. Demographical and laboratory findings are shown in Tables Following through examination, otologic and audiometric evaluation, 40 patients with PCOS and 25 healthy controls were included in the final analysis. Two groups were comparable with regard to age and BMI. The mean age was 23.8  ±  4.6 years in PCOS group and 24.6  ±  4.8 years in control group. The mean BMIs were 25.9 ± 4.8 kg/mA significant difference in LH, LH/FSH, Testosterone, CRP, fasting glucose, fasting insulin, and HOMA index was observed among the two groups .The hearing thresholds for the left and right ears are described in To the best of our knowledge, this is the first study evaluating extended high frequency hearing loss in women with PCOS compared to healthy women. Routine audiometry is restricted to 125–8000 Hz frequencies. However, in diseases with endothelial damage, high frequencies are mostly affected in early stages. High frequency audiometry was introduced into clinical practice in 1960s [P < 0.05). A meta-analysis of the most comparable studies indicates that circulating CRP is elevated in PCOS reflective of the chronic low-grade inflammation present in the disorder. They also found that elevated circulating CRP in PCOS is independent of obesity [In the present study, HOMA-I, CRP, LH, LH/FSH, testosterone, fasting glucose, and insulin levels were higher in women with PCOS. CRP is a circulating marker of the proinflammatory state in PCOS as evidenced by the 2-fold elevation in circulating CRP compared to controls. Similarly, in our study CRP was significantly higher in PCOS patients  hearing loss in PCOS patients. They attributed this to the hyperandrogenism seen in the syndrome . In the Despite certain limitations of our study, the results might be as preliminary findings for designing future studies on larger populations. A limitation of this study is small population, but strength of this study is evaluating the EHFA in all participants.High frequencies are more sensitive to vascular damage caused by the disease. Insulin resistance, hyperandrogenemia, and elevated serum CRP as an inflammatory marker in PCOS could be the cause of hearing loss in especially extended high frequencies. Our data suggest that HFA and EHFA are more efficient in detecting early hearing loss compared to pure tone audiometry. Further studies are needed to help elucidate the mechanism behind hearing impairment in association with PCOS and to see whether the impairment of EHFA in these cases are progressive. It might be possible to prevent progression of hearing impairment by revealing underlying factors.


Protein refolding is an important process to recover active recombinant proteins from inclusion bodies. Refolding by simple dilution, dialysis and on-column refolding methods are the most common techniques reported in the literature. However, the refolding process is time-consuming and laborious due to the variability of the behavior of each protein and requires a great deal of trial-and-error to achieve success. Hence, there is a need for automation to make the whole process as convenient as possible. In this study, we invented an automatic apparatus that integrated three refolding techniques: varying dilution, dialysis and on-column refolding. We demonstrated the effectiveness of this technology by varying the flow rates of the dilution buffer into the denatured protein and testing different refolding methods. We carried out different refolding methods on this apparatus: a combination of dilution and dialysis for human stromal cell-derived factor 1 (SDF-1/CXCL12) and thioredoxin fused-human artemin protein (Trx-ARTN); dilution refolding for thioredoxin fused-human insulin-like growth factor I protein (Trx-IGF1) and enhanced fluorescent protein (EGFP); and on-column refolding for bovine serum albumin (BSA). The protein refolding processes of these five proteins were preliminarily optimized using the slowly descending denaturants (or additives) method. Using this strategy of decreasing denaturants concentration, the efficiency of protein refolding was found to produce higher quantities of native protein. The standard refolding apparatus configuration can support different operations for different applications; it is not limited to simple dilution, dialysis and on-column refolding techniques. Refolding by slowly decreasing denaturants concentration, followed by concentration or purification on-column, may be a useful strategy for rapid and efficient recovery of active proteins from inclusion bodies. An automatic refolding apparatus employing this flexible strategy may provide a powerful tool for preparative scale protein production. E. coli, due to their low cost, high productivity, and ease of use An explosion in the field of structural genomics and protein expression has greatly increased our knowledge of how to manipulate proteins According to the Refold Home dataset, the most common methods reported in the literature regarding protein refolding include dilution, step-wise dialysis and on-column techniques The recovery of active recombinant proteins from inclusion bodies is also a time consuming and laborious process, including multiple steps such as solution preparation, protein concentration, purification and trials of refolding techniques. Due to the inconsistencies and difficulties of the protein refolding process, there is a need for an automated large-scale refolding apparatus that integrated dilution, dialysis and on-column refolding, thereby reducing the need for experimenter’s time.An effective refolding apparatus system configuration should support multiple operations for different applications, including refolding condition variables, such as total protein concentration, denaturants concentration, refolding time, fluid path and refolding methods. The refolding apparatus can be set and maintained throughout the refolding process by adjusting the flow rates of the buffer, the denatured protein suspension, or the fluid path. In this study, we integrated various refolding approaches followed by protein concentration and purification on column. Different methods were carried out to evaluate the performance of this refolding apparatus: a combination of dilution and dialysis for human stromal cell-derived factor 1 (SDF-1/CXCL12) and thioredoxin-human artemin fusion protein (Trx-ARTN); dilution refolding for thioredoxin-human insulin-like growth factor I fusion protein (Trx-IGF1) and enhanced fluorescent protein (EGFP); and on-column refolding for BSA. The refolding processes of these five proteins were preliminary optimized using a strategy of slowly descending denaturant concentration.The refolding apparatus consisted of a compact separation unit and a control system unit with a laptop computer running the system control software. The compact separation unit utilized three refolding techniques that were carried out, respectively, by a dilution module, an ultrafiltration module and an on-column refolding module. In the fluid path of the refolding apparatus, the three refolding modules were coupled with a refolding reservoir. The dilution module consisted of a 4-channel gradient valve (V1), a pump (P1) and a refolding reservoir. Using pump (P1) and 4-channel gradient valve (V1), the denatured, unfolded and fully reduced protein samples in a concentrated denaturant solution were directly placed into the refolding reservoir that contained a large volume of buffer.The ultrafiltration module was attached to the refolding reservoir to aid in the refolding by dialysis. The ultrafiltration module contained two 3-way valves (V2 and V3), a pump (P2) and an ultrafiltration device. Each 3-way valve was assembled using two 2-way valves. In the dialysis refolding, denatured, unfolded and fully reduced protein samples in concentrated denaturant solution were held in the refolding reservoir. A renaturation buffer was continuously fed into the refolding reservoir with pump (P1); simultaneously, the denaturants were continuously filtered and removed using an ultrafiltration membrane. The retentate with proteins was returned to the refolding reservoir. Conventionally, the dialysis bag that contained the denatured protein was immersed in a large volume of refolding solvent in a large reservoir. In this study, the denatured protein was held in the reservoir, not a dialysis bag, so refolding efficiency was controlled easily by both adding solvent additives directly and manipulating the folding pathway, as previously reported 1 is the concentration of denaturants in the feed buffer added into the refolding reservoir, F1 is the flow rate of the feed buffer added into the refolding reservoir, F2 is the flow rate of the removed filtrate, C2 is the concentration of denaturants in the removed filtrate as described in 0 is the initial volume of buffer in the refolding reservoir. If C1 = 0 and F1 = F2, the volume of the buffer in the refolding reservoir was constant, and the change in concentration of denaturants  over time can be calculated as follows:In In To expand the versatility of the apparatus, components for refolding on-column or on-line column capture of refolded proteins were added to the apparatus. These improvements included three FlowDir valves , on-line filters, a 4-channel gradient valve (V4), a Pump (P3), one 2-way valve (V5), a conductivity/UV/pH detector and a series of packed columns. To remove the aggregates formed in the reservoir, on-line filters containing 10 µm and 1 µm filter membranes were added. These filters protected the protein chromatography columns when the soluble protein solution from the reservoir was loaded onto the column. Four different solvents could be chosen using the 4-channel gradient valve (V4), which could serve as the protein purification system. The FlowDir valves (V6 and V7) were five-way valves; at a given setting, two adjacent channels were connected and an axial channel was connected with one of the remaining two channels. The FlowDir valve (V8) was a six-way valve, in which every two adjacent channels were connected. The FlowDir valve (V6) linked pump (P1), reservoir, FlowDir valve (V7) and FlowDir valve (V8). The FlowDir valve (V6) could switch between four positions: (i) linking pump (P1) and the reservoir, or FlowDir valve (V8) and the reservoir; (ii) linking pump (P1) and FlowDir valve (V7); (iii) linking pump (P1) and FlowDir valve (V8); (iv) linking FlowDir valve (V8) and FlowDir valve (V7), or pump (P1) and the reservoir see . The FloWe developed protocols to refold and oxidize SDF-1/CXCL12, Trx-ARTN, and Trx-IGF1 from bacterially expressed inclusion bodies in previous study, whereas the protein refolding efficiency was low (unpublished data). Therefore, we would try to improve the refolding efficiency of these proteins using different refolding techniques on this apparatus. In addition, we chose BSA and EGFP to compare protein refolding efficiency with previously studies. Furthermore, these five proteins have different molecular weight (from 8.9 kDa to 66.4 kDa), pI (acidic or basic) and folded states , and used different denaturants and refolding techniques. In summary, these five proteins can have certain representativeness and are used to demonstrate the advantage and generality of this apparatus  E. coliTo test the quality and efficiency of an optimized refolding process and to compare it with a previous refolding process, we analyzed protein refolding yield and their folding properties. The protein refolding yield was the yield of the total soluble protein compared with the starting amount of denatured proteins. Protein folding properties were used to estimate protein refolding recovery. SDF-1/CXCL12  is a small cytokine belonging to the chemokine family, which has 4 conserved cysteines forming 2 disulfide bonds We developed a protocol to refold and oxidize SDF-1/CXCL12 from bacterially expressed inclusion bodies, which were solubilized using denaturing buffer containing 6 M guanidine hydrochloride. The denatured proteins were slowly added into the refolding buffer containing 0.75 M L-Arg. After 24 h, the refolded proteins were dialyzed against an equilibrium buffer and purified using a CM sepharose resin. Finally, the CM sepharose eluate was analyzed using a Superdex-75 column. The chemokine SDF-1/CXCL12 is a monomer at low protein concentrations, but at concentrations above 200 µM, the observed mass of SDF-1/CXCL12 corresponded to a dimer 2O and refolding buffer were used to rinse the 4-way gradient valve (V1) and pump (P1) into the reservoir through the Dirflow valve (V6) and then pumped out by pump (P2) through 3-way valve (V2). Then, a defined amount of refolding buffer was introduced into the reservoir, and denatured SDF-1/CXCL12 was slowly added into the refolding buffer. Before the end of the refolding time in the solution containing L-Arg, equilibrium buffer was used to rinse 4-way gradient valve (V1), pump (P1), the detector  and the ultrafiltration membrane through Dirflow valve (V6) and Dirflow valve (V8). Afterward, equilibrium buffer was continuously fed into the reservoir by pump (P1). At the same time, the SDF-1/CXCL12 solution was pumped through the ultrafiltration membrane by pump (P2) and returned to the reservoir through Dirflow valve (V8) and Dirflow valve (V6). After 12 h of dialysis, equilibrium buffer was used to rinse 4-way gradient valve (V1), pump (P1) and the detector through Dirflow valve (V6), Dirflow valve (V7), Dirflow valve (V8), and the off-column tubing. Then, equilibrium buffer was introduced into the column to equilibrate the column by adjusting Dirflow valve (V7). Next, soluble SDF-1/CXCL12 solution can be directly loaded onto the column by pump (P2) through Dirflow valve (V8), Dirflow valve (V6), Dirflow valve (V7) and the detector. The refolded SDF-1/CXCL12 protein was eluted with a linear gradient from 4-way gradient valve (V1). In the last step, the washing buffer was pumped into the system to wash the entire system. An animation of the entire refolding process of SDF-1/CXCL12 was supplied in the supporting information  concentration. The optimized refolding process used a refolding buffer that contained 0.75 M L-Arg and was slowly added into the 6 M GdnHCl denatured protein solution. The initial protein concentration was 1.0 mg/ml. This reverse-dilution method generated a linear decreasing concentration of denaturant (GdnHCl)to 0.6 M at a rate of 4.2 mM/min, a linear decreasing protein concentration to 0.1 mg/ml at a rate of 0.042 mg/h, and a linear increasing L-Arg concentration to 0.7 M at a rate of 0.53 mM/min. Otherwise the refolding process was the same as described above. Using this optimized refolding process, 10.5 mg of ∼92% pure and soluble SDF-1/CXCL12 was eluted from CM sepharose resin from 30 mg of ∼60% pure denatured proteins. The animation of the entire refolding process of SDF-1/CXCL12 was supplied in supporting information  from 18 mg of ∼75% pure denatured protein by a combination of dilution and dialysis  of soluble Trx-IGF1obtained from 30.1 mg of ∼70% pure denatured protein by rapid dilution. At each stage during the refolding and dialysis and Ni-column purification and concentration steps, there were almost the same refolding yields of soluble protein from both protein refolding processes  was the key to integrating the dilution unit, dialysis unit and on-column unit within one apparatus. Therefore, we could combine two or three of these units together to complement each other in order to developing protein refolding techniques and processes. In this study of refolding methods, we carried out a combination of dilution and dialysis for SDF-1/CXCL12 and Trx-ARTN, dilution refolding for Trx-IGF1 and EGFP and on-column refolding for BSA, followed by protein concentration and purification on column.We also tried to optimize the protein refolding process by means of the descending denaturant concentration method followed by protein concentration and purification on column. A descending denaturant concentration gradient was generated using a reverse-dilution method and a continuous dialysis method. Using this strategy, the efficiency of protein refolding was found to produce a higher quantity of soluble and correctly folded protein than that of previous refolding processes. In the refolding of SDF-1/CXCL12, a combination of reverse-dilution and dialysis method produced almost double amount of soluble protein than a combination of dilution and dialysis method. Soluble and purified SDF-1/CXCL12 from both refolding processes could self-associate and had similar main CD features of secondary structures as previously reported. Although in the refolding of Trx-ARTN a combination of reverse-dilution and dialysis method could not change the protein refolding yield of soluble protein, indeed it could increase the protein refolding recovery and produce almost three-fold the amount of intermolecular disulphide bridge formed homodimer than a combination of dilution and dialysis method. In contrast with previous refolding processes, the protein refolding yields of soluble proteins of Trx-IGF1 and BSA were not improved obviously as the same to Trx-ARTN using a slowly descending denaturant concentration method, whereas the protein refolding recovery of correctly folded protein was increased. In the refolding of Trx-IGF1 using reverse-dilution method, protein aggregation was suppressed to some extent and the yield of monomer was increased. The protein aggregation in the refolding of Trx-IGF1 would be significantly suppressed again using the refolding buffer containing L-Arg as identified in the literature The reason that the slowly descending denaturant concentration method could help protein refolding can be explained as follows. Nascent polypeptides pass through structural intermediates before the native state is reached. Folding intermediates typically expose hydrophobic residues that are normally buried in the native state. These exposed hydrophobic surfaces are prone to trigger aggregation of proteins In this apparatus, the change rate of concentration of denaturants (or additives) can be manipulated to control the protein folding pathway in order to improve the refolding efficiency. Other factors affecting protein folding, such as redox environment, ionic strength, and pH, would be monitored and manipulated on this refolding apparatus. This refolding apparatus system has a great potential to support multiple operations for different applications, not limited to these studies in this paper. In the refolding of MMP-12 In conclusion, we integrated three refolding techniques into our apparatus: varying dilution, dialysis and on-column refolding. This automatic refolding apparatus can be used for convenience and as a means of developing protein refolding techniques.NdeI and Xhol sites of pET30a expression vector (Novagen), and the genes encoding ARTN and IGF1 were cloned into the KpnI and Xhol sites of pET32m expression vector (Novagen). The plasmids were transferred into BL21 (DE3) CodonPlus strain (Stratagene). SDF-1/CXCL12, Trx-ARTN, Trx-IGF1 and EGFP were expressed in E. coli as inclusion bodies. The cells were grown in 1 liter of LB medium at 37°C until the absorbance at 600 nm (OD600) reached 0.7–0.8. At this time, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to 1 mM to induce protein expression at 37°C for another 4 h. The cells were harvested by centrifugation at 6,000 g at 4°C for 20 min, and washed twice with 150 ml of cell washing buffer , centrifuged . The cells were lysed with 20 ml of cell lysing buffer  by sonication on ice. The lysate was centrifuged . The pellets were washed twice with 150 ml of inclusion bodies washing buffer , the solution was stirred for 1 h, and then centrifuged . To remove Triton X-100, the inclusion bodies were washed two more times with 150 ml of cell washing buffer  and stirred for 1 h. Finally, the pellets were kept at −20°C.The gene encoding EGFP was cloned into pTO-T7 expression vector The isolated inclusion bodies  were dissolved in 10 ml denaturing buffer . The solution was stirred overnight and then centrifuged . The supernatant was collected and pellets were discarded. Denatured Trx-ARTN and Trx-IGF1 were purified by Ni chelating sepharose FF  under denaturing conditions. Denaturing buffer containing 50 mM imidazole was used to wash unbound and impure proteins. The target protein was eluted with denaturing buffer containing 400 mM imidazole. The total protein concentration was measured with the Bradford assay The preparation of inclusion bodies and denatured EGFP protein was described in the Merck iFold™ protein refolding screening system one kit protocol Pure BSA was purchased from Sigma-Aldrich  and dissolved in a denaturing buffer containing 8 M urea as previously reported An animation of the entire refolding process of SDF-1/CXCL12 by the automated refolding apparatus was supplied in the supporting information .2O was used to rinse four inlets  of the 4-way gradient valve (V1) through pump (P1) into the reservoir at a flow rate of 40 ml/min for 1 minute. ddH2O in the reservoir was pumped out through 3-way valve (V2) by pump (P2). The SDF-1/CXCL12 denaturing buffer  and refolding buffer  were used to rinse two inlets  of 4-way gradient valve (V1) through pump (P1) into the reservoir at a flow rate of 20 ml/min for 1 minute. Then the solution in the reservoir was pumped out through 3-way valve (V2) by pump (P2).In the preparation stage, FlowDir valve (V6) was set to link pump (P1) and the reservoir. ddHIn the refolding and purification stage, 300 ml of refolding buffer was added into the reservoir through pump (P1) at a flow rate of 100 ml/min. Then, 30 ml of denaturing protein (A1) at 1.0 mg/ml was slowly injected into the reservoir at 1 ml/min. 2.5 ml of refolding buffer was used to push the residual denatured protein into the reservoir at 1 ml/min. After 24 h, FlowDir valve (V6) was set to link pump (P1) and DirFlow valve (V8). Equilibrium buffer  was used to rinse inlet A3 of the 4-way gradient valve (V1), pump (P1), the detector and ultrafiltration device throughout 3-way valve (V2) at a flow rate of 10 ml/min for 5 min. FlowDir valve (V6) was set to link pump (P1) and the reservoir, or the reservoir and DirFlow valve (V8). Equilibrium buffer was continuously added into the reservoir by pump (P1) at a flow rate of 2 ml/min. The outlet of the reservoir provided a steady continuous flow of fluid at 50 ml/min by pump (P2) into the ultrafiltration device. The retained solution returned to the reservoir through the detector. The flow rate of the filtrate was set at 2 ml/min. After 12 h of dialysis, FlowDir valve (V6) was set to link pump (P1) and FlowDir valve (V7), the FlowDir valve (V7) was set to link FlowDir valve (V6) and off-column way, and the detector was set within the purification system. Equilibrium buffer and eluent  were used to rinse the purification system at a flow rate of 20 ml/min for 1 minute. Afterwards, FlowDir valve (V7) was set to link FlowDir valve (V6) and the CM-sepharose fast flow column (5 ml). Equilibrium buffer was used to equilibrate the column at a flow rate of 2 ml/min for 5 column volumes (CVs). FlowDir valve (V6) was set to link pump (P1) and the reservoir, or FlowDir valve (V8) and FlowDir valve (V7). Refolded SDF-1/CXCL12 in the reservoir was directly loaded onto the CM-sepharose fast flow column by pump (P2) at a flow rate of 5 ml/min. The residual sample in the reservoir and ultrafiltration device was pushed into the column with equilibrium buffer by pump (P1) at a flow rate of 2 ml/min. The column was washed with equilibrium buffer until the UV absorbance at 280 nm returned to a stable baseline. The refolded SDF-1/CXCL12 protein was eluted with a 20 CV linear gradient from 0% to 100% eluent at a flow rate of 2 ml/min.2O was used to wash the entire system. FlowDir valve (V7) was set to link FlowDir valve (V6) and the off-column way. The detector was set within the purification system and ddH2O  was used to wash the purification system at a flow rate of 20 ml/min for 1 minute. After that, FlowDir valve (V7) was set to link FlowDir valve (V6) and the column. ddH2O was used to wash the column. FlowDir valve (V6) was set to link pump (P1) and the reservoir. ddH2O was used to wash the reservoir at a flow rate of 40 ml/min for 5 min and ddH2O in the reservoir was pumped out through 3-way valve (V2) by pump (P2). FlowDir valve (V6) was set to link pump (P1) and DirFlow valve (V8). ddH2O was used to wash the ultrafiltration device throughout Out(1) at a flow rate of 40 ml/min for 5 min.In the final stage, ddHThe refolded SDF-1/CXCL12 eluted from the CM-sepharose fast flow column was concentrated using an YM-3 membrane (Amicon) and purified by a Superdex-75 10/300 column  on an AKTApurifier UPC100.Two different protein concentrations of purified SDF-1/CXCL12 (1.2 and 8 mg/ml) were analyzed by a Superdex-75 10/300 column.The animation of the entire refolding process of SDF-1/CXCL12 by the automated refolding apparatus was supplied in the supporting information .The preparation stage was the same as described in the section of “Batch refolding of SDF-1/CXCL12 by dilution and dialysis combination refolding method.”In the refolding and purification stage, 30 ml of denatured protein at 1.0 mg/ml was added into the reservoir through pump (P1) at a flow rate of 2 ml/min. Then, 300 ml of refolding buffer (A2) was slowly injected into the reservoir at a flow rate of 0.23 ml/min for approximately 21 h. The rest of the procedure was the same as described in the section of “Batch refolding of SDF-1/CXCL12 by dilution and dialysis combination refolding method”.The refolded SDF-1/CXCL12 eluted from CM-sepharose fast flow column was concentrated using a YM-3 membrane (Amicon) and purified by a Superdex-75 10/300 column  on an AKTApurifier UPC100.Two different protein concentrations of purified SDF-1/CXCL12 (1.6 and 8 mg/ml) in were analyzed by a Superdex-75 10/300 column.The animation of the entire refolding process of Trx-ARTN by the automated refolding apparatus was the same as that of SDF-1/CXCL12.All operations were the same as described in the section of “Batch refolding of SDF-1/CXCL12 by dilution and dialysis combination refolding method”. 45 ml of denatured protein at 0.49 mg/ml was slowly added into 450 ml of refolding buffer . The equilibrium buffer was 20 mM TrisHCl, 1 mM EDTA, pH 8.0. The eluent was 20 mM TrisHCl, 1 mM EDTA, 1.0 M NaCl, pH 8.0.The refolded Trx-ARTN eluted from the CM-sepharose fast flow column was analyzed by reduced SDS-PAGE and non-reduced SDS-PAGE.The animation of the entire refolding process of Trx-ARTN by the automated refolding apparatus was the same as that of SDF-1/CXCL12.All operations were the same as described in the section of “Batch refolding of SDF-1/CXCL12 by reverse-dilution and dialysis combination refolding method”. 370 ml of refolding buffer was slowly added into 37 ml of denatured protein at 0.64 mg/ml.The refolded Trx-ARTN eluted from the CM-sepharose fast flow column was analyzed by reduced SDS-PAGE and non-reduced SDS-PAGE.The animation of the entire refolding process of Trx-IGF1 by the automated refolding apparatus was shown in the supporting information (Video: Trx-IGF1 refolding process of dilution.mp4).The preparation stage was the same as described in the section of “Batch refolding of SDF-1/CXCL12 by dilution and dialysis combination refolding method”. The ultrafiltration device was removed from the refolding apparatus.In the refolding and purification stage, 450 ml of refolding buffer  was added into the reservoir through pump (P1) at a flow rate of 100 ml/min. Then, 43 ml of denatured protein (A1) at 0.7 mg/ml was slowly injected into the reservoir at 1 ml/min. 2.5 ml of refolding buffer was used to push the residual denaturing protein into the reservoir at 1 ml/min. After 24 h, FlowDir valve (V6) was set to link pump (P1) and DirFlow valve (V8). Equilibrium buffer  was used to rinse inlet A3 of the 4-way gradient valve (V1), pump (P1), detector and tubes in the dialysis unit throughout Out(1) at a flow rate of 10 ml/min for 5 min. FlowDir valve (V6) was set to link pump (P1) and FlowDir valve (V7). FlowDir valve (V7) was set to link FlowDir valve (V6) and off-column way. The detector was set within the purification system and equilibrium buffer and eluent  were used to rinse the purification system at a flow rate of 20 ml/min for 1 min. After that, FlowDir valve (V7) was set to link FlowDir valve (V6) and the Ni-chelating fast flow column (20 ml). Equilibrium buffer was used to equilibrate the column at a flow rate of 4 ml/min for 5 column volumes (CVs). FlowDir valve (V6) was set to link pump (P1) and the reservoir, or the FlowDir valve (V8) and the FlowDir valve (V7). Refolded Trx-IGF1 in the reservoir was directly loaded onto the Ni-chelating sepharose fast flow column by pump (P2) at a flow rate of 10 ml/min. The residual sample in the reservoir and ultrafiltration device was pushed into the column with equilibrium buffer by pump (P1), at a flow rate of 4 ml/min. The column was washed with 5% eluent until the UV absorbance at 280 nm returned to a stable baseline. The refolded Trx-IGF1 protein was eluted with 50% eluent at a flow rate of 4 ml/min.The last stage was the same as described in the section of “Batch refolding of SDF-1/CXCL12 by dilution and dialysis combination refolding method”.The refolded Trx-IGF1 eluted from Ni-column was concentrated using a YM-10 membrane (Amicon) and purified by a Superdex-200 10/300 column. The monomeric protein fractions were collected and concentrated using a YM-10 membrane (Amicon). The concentrated protein solution was purified again with a Superdex-200 10/300 column. The monomeric and pure Trx-IGF1 was collected and analyzed by a Superdex-200 10/300 column.The animation of the entire refolding process of Trx-IGF1 by the automated refolding apparatus was shown in the supporting information (Video: Trx-IGF1 refolding process of reverse-dilution.mp4).The preparation stage was the same as described in the section of “Batch refolding of SDF-1/CXCL12 by dilution and dialysis combination refolding method”. The ultrafiltration device was removed from the refolding apparatus.In the refolding and purification stage, 50 ml of denatured protein (A1) at 0.7 mg/ml was added into the reservoir through pump (P1) at a flow rate of 2 ml/min, and then 450 ml of refolding buffer was slowly injected into the reservoir at 0.35 ml/min. After 24 h, FlowDir valve (V6) was set to link pump (P1) and DirFlow valve (V8). Equilibrium buffer was used to rinse inlet A3 of the 4-way gradient valve (V1), pump (P1), the detector and tubes in the dialysis unit throughout Out(1) at a flow rate of 10 ml/min for 1 min. FlowDir valve (V6) was set to link pump (P1) and FlowDir valve (V7). FlowDir valve (V7) was set to link FlowDir valve (V6) and off-column way. The detector was set within the purification system, and equilibrium buffer and eluent were used to rinse the purification system at a flow rate of 20 ml/min for 1 min. After that, FlowDir valve (V7) was set to link FlowDir valve (V6) and the Ni-chelating sepharose fast flow column (20 ml). Equilibrium buffer was used to equilibrate the column at a flow rate of 4 ml/min for 5 column volumes (CVs). FlowDir valve (V6) was set to link pump (P1) and the reservoir, or the reservoir and DirFlow valve (V8). Refolded Trx-IGF1 solution was used to rinse pump (P2) and was returned to the reservoir. FlowDir valve (V6) was set to link pump (P1) and the reservoir, or the FlowDir valve (V8) and the FlowDir valve (V7). Refolded Trx-IGF1 in the reservoir was directly loaded onto the Ni-chelating sepharose fast flow column by pump (P2) at a flow rate of 10 ml/min. The residual sample in the reservoir and ultrafiltration device was pushed into the column with equilibrium buffer by pump (P1) at a flow rate of 4 ml/min. The column was washed with 5% eluent until the UV absorbance at 280 nm returned to a stable baseline. The refolded Trx-IGF1 protein was eluted with 50% eluent at a flow rate of 4 ml/min.The last stage was the same as described in the section of “Batch refolding of SDF-1/CXCL12 by dilution/dialysis combination refolding method”.The refolded Trx-IGF1 eluted from Ni-column was concentrated using a YM-10 membrane (Amicon) and purified using a Superdex-200 10/300 column. The monomeric protein fractions were collected and concentrated using a YM-10 membrane (Amicon). The concentrated protein solution was purified again using a Superdex-200 10/300 column. The monomeric and pure Trx-IGF1 was collected and analyzed by a Superdex-200 10/300 column.The animation of the entire refolding process of BSA by the automated refolding apparatus was shown in supporting information (video: BSA refolding process of on-column.mp4).2O was used to rinse four inlets  of the 4-way gradient valve (V4) through pump (P3) at a flow rate of 40 ml/min for 1 min. FlowDir valve (V7) was set to link pump (P3) and the HiTrap Q-sepharose FF column (1 ml). ddH2O was used to rinse the column at a flow rate of 1 ml/min for 5 CVs. FlowDir valve (V7) was set to link pump (P3) and the off-column way. Denaturing buffer  and refolding buffer  were used to rinse the purification system at a flow rate of 20 ml/min for 1 min. FlowDir valve (V7) was set to link pump (P3) and the HiTrap Q-sepharose FF column. Denaturing buffer was used to equilibrate the column at a flow rate of 1 ml/min for 10 CVs.In the preparation stage, FlowDir valve (V7) was set to link pump (P3) and the off-column way. The detector was set within the purification system, and ddHIn the refolding stage, 4 ml of denatured protein (B4) at 0.5 mg/ml was injected onto the column through pump (P3) at a flow rate of 1 ml/min. Then, 2.5 ml of denaturing buffer (B4) was used to push the residual denatured protein, aided by pump (P3) onto the column. The column was washed with 5 CVs to remove unbound protein. Refolding was initiated by switching from denaturing buffer to refolding buffer over 5 CVs. Then, the flow rate was set to zero, and the protein was left on the column to incubate for up to 40 h. FlowDir valve (V7) was set to link pump (P3) and the off-column way. Equilibrium buffer and eluent were used to rinse the purification system at a flow rate of 20 ml/min for 1 min. FlowDir valve (V7) was set to link pump (P3) and the HiTrap Q-sepharose FF column. Equilibrium buffer was used to wash the column with 5 CVs. Elution was then initiated by a salt gradient. A 20 CV linear gradient from 100% TrisHCl buffer containing no NaCl to that containing 1 M NaCl was applied to elute protein from the column.2O was used to wash four inlets  of the 4-way gradient valve (V4) through pump (P3) at a flow rate of 40 ml/min for 1 min. FlowDir valve (V7) was set to link pump (P3) and the HiTrap Q-sepharose FF column. ddH2O was used to rinse the column at a flow rate of 1 ml/min for 5 CVs.In the last stage, FlowDir valve (V7) was set to link pump (P3) and the off-column way. The detector was set within the purification system and ddHProtein-containing fractions were pooled and analyzed by a CN reverse-phase column  on an Agilent HP1100 using 40% acetonitrile solution at flow rate of 1.0 ml/min, to determine the concentrations of refolded and partially folded protein.The animation of the entire refolding process of BSA by the automated refolding apparatus was shown in supporting information .The preparation stage was the same as described in the section of “Batch refolding of SDF-1/CXCL12 by dilution/dialysis combination refolding method”. The ultrafiltration device was removed from the refolding apparatus.In the refolding and purification stage, 110 ml of denatured protein (A4) at 0.1 mg/ml was added into the reservoir through pump (P1) at a flow rate of 10 ml/min. Then, approximately 480 ml of refolding buffer (A3) was continuously added into the reservoir at a flow rate of 0.2 ml/min for approximately 40 h. The outlet of the reservoir provided a steady continuous flow of fluid at 10 ml/min by pump (P2) into the ultrafiltration device, and the retained solution returned to the reservoir through detector. The flow rate of the filtrate was set at 0.2 ml/min. The urea concentration was decreased to 0.1 mM. The following work was the same as described in the section of “Batch refolding of SDF-1/CXCL12 by dilution/dialysis combination refolding method”.Protein-containing fractions were pooled and analyzed by RP-HPLC to determine the concentrations of refolded and partially folded proteins.The animation of the entire refolding process of EGFP by the automated refolding apparatus was shown in supporting information (video: EGFP refolding process of dilution.mp4).2O was used to rinse two inlets  of 4-way gradient valve (V1) through pump (P1) into the reservoir at a flow rate of 20 ml/min for 1 min. ddH2O in the reservoir was pumped out through 3-way valve (V2) by pump (P2). EGFP denaturing buffer  and refolding buffer  were used to rinse two inlets  of the 4-way gradient valve (V1) through pump (P1) into the reservoir at a flow rate of 20 ml/min for 1 min. Then, the solution in the reservoir was pumped out through 3-way valve (V2) by pump (P2).In the preparation stage, FlowDir valve (V6) was set to link pump (P1) and the reservoir. ddHIn the refolding and purification stage, 500 ml of refolding buffer was added into the reservoir through pump (P1) at a flow rate of 100 ml/min, and then 50 ml of denaturing protein containing 0.06% N-lauroylsarcosine (A4) at 1.0 mg/ml was slowly injected into the reservoir at 1 ml/min. 2.5 ml of refolding buffer was used to push the residual denaturing protein into the reservoir at 1 ml/min. After 8 h (overnight), refolded protein in the reservoir was pumped out through Out(1) by pump (P2). Residual refolded protein in the reservoir was washed out by refolding buffer.2O was used to wash the reservoir and was then pumped out by pump (P2).In the last stage, ddHThe animation of the entire refolding process of EGFP by the automated refolding apparatus was shown in supporting information .The preparation stage was the same as described in the section of “Batch refolding of BSA by continuous dialysis refolding method”.In the refolding and purification stage, 500 ml of denatured protein containing 0.06% N-lauroylsarcosine (A4) at 0.1 mg/ml was added into the reservoir through pump (P1) at a flow rate of 20 ml/min. Then, approximately 2000 ml of refolding buffer containing 1.0 mM methyl-β-D-cyclodextrin (A3) was continuously added into the reservoir at a flow rate of 4.2 ml/min for 8 h. The outlet of the reservoir provided a steady continuous flow of fluid at 50 ml/min by pump (P2) into the ultrafiltration device, and the retained solution returned to the reservoir through the detector. The flow rate of the filtrate was set at 4.2 ml/min. After 8 h (overnight), refolded protein in the reservoir was pumped out through Out(3) by pump (P2). Residual refolded protein in the reservoir was washed out by refolding buffer.2O was used to wash the reservoir and the ultrafiltration membrane and was then pumped out by pump (P2).In the last stage, ddHTo investigate the effects on EGFP refolding using direct dilution or continuous dialysis refolding techniques, the fluorescence spectra of four EGFP samples were analyzed. These samples included unfolded EGFP (negative control), soluble expressed EGFP, and two refolded EGFP from inclusion bodies and were adjusted to concentration of 1.0 mg/ml. Excitation was set at 450 nm and emission spectra at 400–650 nm were recorded using a fluorescent spectrometer SpectraMaxM5 (Molecular Devices Inc.). Slit width was set at 2.5/1 nm.2HPO4, pH7.4) at 4°C. CD spectra were measured at 22°C under a nitrogen flow on J-715 spectropolarimeter (Jasco) equipped with a 1 mm path-length cuvette using phosphate buffer as a control. CD spectra were recorded from190 to 250 nm with a step size of 0.1 nm, a bandwidth of 1 nm. The photomultiplier voltage read never exceeded 600 V in the spectral regions. Each spectrum was averaged from five measurements.Purified proteins were dialyzed against phosphate buffer  has 5 ways used for linking pump (P1), the reservoir, FlowDir valve (V8) and FlowDir valve (V7), which could switch between four positions: (A) linking pump (P1) and the reservoir, or FlowDir valve (V8) and the reservoir; (B) linking pump (P1) and FlowDir valve (V7); (C) linking pump (P1) and FlowDir valve (V8); (D) linking FlowDir valve (V8) and FlowDir valve (V7), or pump (P1) and the reservoir.(TIF)Click here for additional data file.Figure S2FlowDir valve (V7) has 4 ways used for linking pump (P3), FlowDir valve (V6), the off-column way and the column, which could switch between three positions: (A) linking FlowDir valve (V6) and the column, or pump (P3) and the off-column way; (B) linking FlowDir valve (V6) and the off-column way; (C) linking pump (P3) and the column.(TIF)Click here for additional data file.Figure S3FlowDir valve (V8) has two switch positions: the detector connected with FlowDir valve (V6) (A) or the column (B).(TIF)Click here for additional data file.Figure S4The change of concentration of urea in the reservoir with time in the MMP-12 refolding using a continuous dialysis process (solid line) and an improved continuous dialysis process (dotted line).(TIF)Click here for additional data file.Figure S5Calibration curve of protein standards of the Superdex-75 10/300 HR column.(TIF)Click here for additional data file.Figure S6Calibration curve of protein standards of the Superdex-200 10/300 HR column.(TIF)Click here for additional data file.Table S1An overview of information of five proteins.(DOC)Click here for additional data file.Video S1CXCL12 refolding process of dilution and dialysis combination.mp4.(MP4)Click here for additional data file.Video S2CXCL12 refolding process of reverse-dilution and dialysis combiantion.mp4.(MP4)Click here for additional data file.Video S3Trx-IGF1 refolding process of dilution.mp4.(MP4)Click here for additional data file.Video S4Trx-IGF1 refolding process of reverse-dilution.mp4.(MP4)Click here for additional data file.Video S5BSA refolding process of on-column.mp4.(MP4)Click here for additional data file.Video S6BSA refolding process of continuous dialysis.mp4.(MP4)Click here for additional data file.Video S7EGFP refolding process of dilution.mp4.(MP4)Click here for additional data file.Video S8EGFP refolding process of continuous dialysis.mp4.(MP4)Click here for additional data file.


Granulin-epithelin precursor (GEP) has previously been reported to controlcancer growth, invasion, chemo-resistance, and served as novel therapeutictarget for cancer treatment. However, the nature and characteristics of GEPinteracting partner remain unclear. The present study aims to identify andcharacterize the novel predominant interacting partner of GEP using co-immunoprecipitationand mass spectrometry.Specific anti-GEP monoclonal antibody was used to capture GEP and its interactingpartner from the protein extract of the liver cancer cells Hep3B. The precipitatedproteins were analyzed by SDS-PAGE, followed by mass spectrometry and theprotein identity was demonstrated to be tropomyosin 3 (TPM3). The interactionhas been validated in additional cell models using anti-TPM3 antibody andimmunoblot to confirm GEP as the interacting partner. GEP and TPM3 expressionswere then examined by real-time quantitative RT-PCR in clinical samples, andtheir transcript levels were significantly correlated. Elevated TPM3 levelswere observed in liver cancer compared with the adjacent non-tumorous liver,and patients with elevated TPM3 levels were shown to have poor recurrence-freesurvival. Protein expression of GEP and TPM3 was observed only in the cytoplasmof liver cancer cells by immunohistochemical staining.TPM3 is an interacting partner of GEP and may play an important role inhepatocarcinogenesis. Hepatocellular carcinoma (HCC) is a malignant neoplasm of hepatocytes andit accounts for more than 80% of primary liver cancers Several important intracellular signaling pathways including the mitogen-activatedprotein kinases comprising the ERK, JNK and p38 have been recognized to beinvolved in hepatocarcinogenesis Our earlier cDNA microarray study revealed differential gene expressionpatterns in HCC and non-tumor liver tissues To further understand the GEP signaling mechanism, the present study aimsto identify its novel predominant interacting partners. Proteins that interactwith GEP were examined using co-immunoprecipitation and mass-spectrometry.The GEP interacting protein had been further examined in additional cell linesand clinical samples using western blot, immunohistochemistry and real-timequantitative PCR.Anti-GEP monoclonal antibody raised against the GEP carboxyl-terminus wasused for immunoprecipitation 2. GEP full-length cDNA andanti-sense fragment construct were transfected into the liver cancer cellsto obtain stable transfectants for GEP overexpression and GEP suppression,respectively. Stable clones were selected by G418 as described previously Two human liver cancer cell lines, Hep3B and HepG2 , were used in the immunoprecipitation experiments.Hep3B was derived from an 8 years old juvenile patient, whereas HepG2 wasderived from a 15 years old adolescent patient. The two cell lines were grownin AMEM medium containing 10% fetal bovine serum (FBS) and L-glutaminesupplement at 37°C in 5% COThe studpy protocol was approved by the Institutional Review Board of theUniversity of Hong Kong/Hospital Authority Hong Kong West Cluster (HKU/HAHKW IRB). Patients underwent curative partial hepatectomy for HCC at QueenMary Hospital, Hong Kong, were recruited with written inform consent to thestudy. Total RNA was extracted from snap frozen tissue specimens for mRNAexpression study using real-time quantitative PCR, whereas formalin-fixedparaffin-embedded tissues were used for histological and immunohistochemicalstudies.Total cell lysates were obtained using NET buffer  in the presence of completeprotease inhibitor (Roche) and 1 mM phenylmethylsulphonyl fluoride . Briefly, cells were first washed by PBS and cell pellet was collectedby trypsinization. The cell pellet was washed using PBS and then NET was addedto resuspend the cells. The lysate was then incubated on ice for 30 minutesand followed by centrifugation at 14,000 x g for 20 minutes at 4°C. Thesupernatant was collected and the concentration of the total protein was determinedusing the BioRad DC Protein Assay Kit . A totalof 400 µg extracted protein lysate was used for each co-immunoprecipitationreaction. The monoclonal anti-GEP antibody which is specific to human GEPwas used to coimmunoprecipitate GEP and its binding partners. Two controlreactions, antibody alone and protein lysate alone, were also included asreference to the non-specific binding of unrelated proteins with the proteinG-Sepharose. Two microgram of monoclonal anti-GEP antibody was incubated with400 µg total protein lysate at 4°C with rotation overnight. Foreach reaction, 100 µl of protein G-Sepharose beads  were washed with 500 µl NET buffer for 3 times. Washedprotein G-Sepharose beads were incubated with the antibody-protein complexesat 4°C with rotation for 1 hour. After incubation, the complexes werebriefly centrifuged and supernatant was discarded. The beads were washed with500 µl ice-cold NET buffer for 5 times to wash any unbounded proteins.The coimmunoprecipition product was then analysed by typical one-dimensionalSDS-PAGE. The immunocomplex-bound protein G-Sepharose beads were resuspendedusing 2×protein buffer . Proteins were denatured at 95°C for 5minutes. The supernatant containing the proteins were separated under denaturingcondition on 10% SDS-PAGE gel and followed by SimplyBlue  staining which is a mass-spectrometry compatible coomassie bluestain. The major differential band observed in the co-immunoprecipitationreaction but absent in the two control reactions  were excised from the gel and further analyzed by mass-spectrometry.The differential protein band was excised from stained gel, and in-geltrypsin digestion was performed as previously described For protein expression on cell lines, aliquot of 20 µg total proteinlysate were separated in 10% SDS-PAGE gel 5′-CCC TGA GAC GGT AAAGAT GCA-3′) and GRN-probe (5′-6FAM CCA CTG CTCTGC CGG CCA CTC MGBNFQ-3′) Real-time quantitative PCR was performed as described previously Protein expression of GEP and TPM3 were investigated by immunohistochemicalstaining on formalin-fixed and paraffin-embedded clinical specimens To identify the GEP interacting proteins, co-immunoprecipitation was performedusing the monoclonal anti-GEP antibody. Liver cancer cells Hep3B expresseda higher GEP levels compared to HepG2 cells, and both levels were significantlyhigher than normal liver tissues. In order to increase the detection efficiencyby using GEP as the ligand, Hep3B cells transfected with GEP full-length construct(Hep3B FL) were used for overexpression of GEP protein. The predominant interactingpartner was identified by separating and comparing the immunoprecipitatedproteins of anti-GEP antibody and those of control setups  by one-dimensional SDS-PAGE. An obvious major differentialband with the size at around 34 kDa was observed only in the reaction lanebut not in the control reaction lanes . The majTo confirm the results obtained from the co-immunoprecipitation and massspectrometry, the interaction between GEP and TPM3 was validated by immunoprecipitationusing anti-TPM3 antibody, then followed with immunoblot detection using anti-GEPantibody in Hep3B FL and HepG2 FL. A band with expected GEP size (90 kDa)was observed only on anti-TPM3 antibody-precipitated protein but not in thecontrol reactions A–B. TheExpression of TPM3 was investigated on a panel of cell lines  and S3. P  = 0.001) (P<0.001)(P  = 0.0496) . The expP<0.001). Increas 0.0496).Protein expression of TPM3 was investigated by immunohistochemical staining,and TPM3 protein was observed only in tumor tissues but not in the non-tumoroustissues  A–D.TheProtein-protein interacting partners are traditionally identified by undertakingthe Western blot approach with the use of specific antibodies. However, thistype of approach is hypothesis-driven, and therefore requires a “goodguess” of the potential interacting partners for choosing the specificantibodies to examine the protein-protein binding. The Western blot approach,hence, is usually not cost effective. Recent advances in proteomic technologiesfor identification of unknown proteins at low quantity have allowed us touse an unbiased and non-hypothesis driven approach to look for novel protein-proteininteracting partners. In this study, we aim to identify the novel predominantinteracting proteins of GEP in HCC. The first phase, identification phase,has been examined with co-immunoprecipitation using anti-GEP antibody to captureGEP and its interacting protein partners, followed with comparative 1D SDS-PAGEanalysis and mass spectrometry analysis. Our result revealed an obvious differentialprotein band as the putative predominant GEP interaction partner, which waslater shown to be TPM3. We subsequently confirmed their interactions and cellularco-localizations by Western blot analysis and histological examination inclinical specimens, respectively. Although we aimed to examine for the predominantbinding partner of GEP in the present study, other minor binding partnersof GEP may exist. Further studies for identifying the minor binding partnersusing nano-LC/MS method are on-going in our laboratories.In the second phase of this study, validation phase, the interaction betweenGEP and TPM3 has been investigated with additional cell models, using anti-TPM3antibody to capture TPM3 and its binding partners, followed by immunoblotto confirm the interaction of GEP with TPM3. Further investigation on clinicalspecimen demonstrated GEP and TPM3 to be overexpressed in HCC compared withnon-tumor liver sample, and the expression levels of GEP and TPM3 were significantlycorrelated. Immunohistochemical staining also revealed that GEP and TPM3 areco-localised in the cytoplasm of HCC cells. The present study consolidatedTPM3 as the interacting partner of GEP.Tropomyosin is an actin-binding protein which exists in both muscle andnon-muscle cells -ALK (anaplastic lymphoma kinase) fusion through chromosome translocation -ALK fusion gene is further investigated to be involvedin transformation, proliferation, invasion and metastasis in anaplastic large-celllymphoma In the present study, we have demonstrated elevated expression of TPM3in HCC. Dysregulation of TPM3 has also been reported in other human diseases.Missense mutation in the TPM3 has been reported to be associated with autosomalnemaline myopathy, a disease characterized by the presence of muscle fibresin the pathognomonic rod bodies In summary, we are the first group to demonstrate that TPM3 is a predominantinteracting partner of GEP in the cytoplasm of HCC cells. Notably, TPM3 hasbeen reported to control migration, invasion and anchorage-independent growthof HCC cells Figure S1Comparison of the reference protein sequences of TPM1-4. Underlinedregions were the MALDI-TOF/TOF-MS analysis of the tryptic peptides matchedto TPM3. Mismatches were highlighted. The 5 isoforms of TPM3 are conservedin the tryptic peptide regions. The 7 isoforms of TPM1, the 2 isoforms ofTPM2 and the 2 isoforms of TPM4 are conserved in the tryptic peptide regionsas shown in the reference sequences.(DOC)Click here for additional data file.FigureS2Suppression of TPM3 by siRNA. Three different siRNAs againstTPM3 were transfected to Hep3B and HepG2 cells respectively. The three controlsincluded the parental cells only (c), cells incubated with lipofectamine only(lipo) and cells mock-transfected with siRNA negative control (NC). TPM3 suppressionby siRNAs decreased the TMP3 mRNA and protein levels but showed insignificanteffect on GEP levels.(DOC)Click here for additional data file.FigureS3Modulation of GEP levels. Suppression of GEP was performedin Hep3B cells with high endogenous GEP levels. Overexpression of GEP wasperformed in HepG2 cells with relatively low endogenous GEP expression. GEPexpression modulations showed minimal effect on TPM3 mRNA levels.(DOC)Click here for additional data file.TextS1Supplementary Materials and Methods.(DOC)Click here for additional data file.