Thus, Soluplus achieved the purpose of improving the flowability and solubility of baicalein phospholipid complexes. The application of Soluplus to phospholipid complexes has great potential.In this study, a novel ternary complex system (TCS) composed of baicalein, phospholipids, and Soluplus was prepared to improve the flowability and dissolution for baicalein phospholipid complex (BPC). TCS was characterized using differential scanning calorimetry (DSC), infrared spectroscopy (IR), powder X-ray diffraction (PXRD), and scanning electron microscopy (SEM). The flowability, solubility, oil–water partition coefficient, in vitro dissolution, and in vivo pharmacokinetics of the system were also evaluated. DSC, IR, PXRD, and SEM data confirmed that the crystal form of baicalein disappeared in BPC and TCS. Furthermore, the angle of repose of TCS of 35° indicated an improvement in flowability, and solubility increased by approximately eight-fold in distilled water when TCS was compared with BPC (41.00 ± 4.89 μg/mL vs. 5.00 ± 0.16 μg/mL). Approximately 91.24% of TCS was released at the end of 60 min in 0.5% SDS (pH = 6.8), which suggested that TCS could improve the dissolution velocity and extent. Moreover, TCS exhibited a considerable enhancement in bioavailability with higher peak plasma concentration (25.55 μg/mL vs. 6.05 μg/mL) and increased AUC The poon-octanol solubility of baicalein was improved in baicalein phospholipid complex (BPC) [Given the phospholipid’s excellent biocompatibility and unique amphiphilicity, a drug–phospholipid complex has been employed as a technique to improve the oral absorption of the drugs which belong to BCS class III and IV ,10,11. Dex (BPC) . NeverthSoluplus B has beeIn the present study, a novel ternary complex system (TCS) composed of baicalein, phospholipids, and Soluplus was prepared to improve the flowability and dissolution in vitro of BPC. Due to ameliorative flowability, the successful development of TCS has created favorable conditions for large-scale production of baicalein in industrial production. Likewise, the improvement in dissolution is also advantageous for the improvement of bioavailability. Based on these findings, we conclude that Soluplus achieved the purpose of improving the flowability and solubility of BPC. The safe and effective excipients incorporation of phospholipid complex provides a new idea for its better development. In our design, TCS was prepared via solvent evaporation with baicalein, phospholipids, and Soluplus. First, TCS was characterized using differential scanning calorimetry (DSC), infrared spectroscopy (IR), powder X-ray diffraction (PXRD), and scanning electron microscopy (SEM). Second, flowability, solubility, oil–water partition coefficient, and dissolution in vitro were evaluated. Finally, the baicalin content was detected in rats after oral administration to further assess bioavailability via High Performance Liquid Chromatography-Electrospray Ionization-Mass Spectrometry/Mass Spectrometry (HPLC-ESI-MS/MS) .Baicalein and baicalin were purchased from Sichuan Weikeqi Biology Technique Co., Ltd. Soluplus was kindly gifted by BASF SE . Icariin was purchased from the China Institute of Pharmaceutical and Biological Products and employed as an internal standard. All other chemical reagents used in the experiments were analytical grade or better. Purified Milli-Q water was utilized in the experiments .The drug and phospholipid (1:2 mass ratio) were used to prepare BPC. In brief, baicalein and phospholipid (phosphatidylcholine) were dissolved in 10 mL of absolute ethanol. The solution was magnetically stirred for 0.5 h and then vacuum dried for 24 h to obtain the solid product, which was stored in a desiccator.The drug and phospholipid (BPC) with Soluplus were weighed at a mass ratio of 1:2:2 and dissolved in 10 mL of absolute ethanol. The first two were mixed and dissolved by magnetic stirring for 0.5 h. Then, the weighed Soluplus was added to the above mixed solution, stirring to dissolve completely. Further stirring for 0.5 h resulted in a Soluplus complex with mass ratios of 1:2:2. The complex was vacuum dried for 24 h to obtain the solid powder, which was then stored in a desiccator.PM was prepared by thoroughly mixing baicalein, phospholipid, and Soluplus (1:2:2 mass ratio) in a mortar for 10 min until a homogenous mixture was obtained. The sample was stored in a desiccator.The thermal properties of Baicalein, BPC, TCS, and PM of TCS were studied using a differential scanning calorimeter  equipped with a thermal analysis system. Dry nitrogen was used as the purge gas (purge 40 mL/min). The right amount of samples of baicalein, BPC, TCS, and PM were weighed into an aluminum pan. The probes were heated at a temperature of 0–300 °C at a rate of 20 °C/min .−1.The IR spectra of baicalein, BPC, TCS, and PM of TCS were obtained using an IR spectrophotometer . Samples were scanned over the wavenumber range of 4000–400 cmThe crystalline state of prepared baicalein, BPC, TCS, and PM of TCS was acquired at room temperature with Cu Kα radiation source at 40 kV and 25 mA via XRD .The granule morphology of baicalein, BPC, TCS, and PM of TCS were determined using a SEM . The samples were sputter-coated  with gold–palladium and then observed at different magnifications.After TCS was added into the hopper, it was allowed to flow uniformly through the funnel until the highest point of the cone. The height (h) and radius (r) of the cone were then measured to calculate the angle of repose (θ).n-octanol (5.0 mL) was added to excess baicalein, BPC, and TCS to determine solubility in the swing bed at 37 °C for 24 h [Water or for 24 h . The liqn-octanol (water saturation) was shaken to balance, 2.0 mL of water (n-octanol saturation) was added. The miscible liquid was then agitated for 24 h, and the concentration in each phase was determined using HPLC after standing for layering. Experiments for each sample were performed in triplicate.After 2.0 mL of baicalein, BPC, or TCS-saturated v/v), and the flow rate was 1.0 mL/min. The injection volume was 10 μL. The method was optimized from the protocol described by Zhang [All samples were analyzed using Waters HPLC with a quaternary pump  on a Diamonsil C18 column (200 mm × 4.6 mm × 5 μm) at 276 nm, which was maintained at 35 °C. The mobile phase was methanol-0.1% formic acid  was used according to appendix XC of Chinese Pharmacopoeia 2010 edition. The samples, which were equivalent to 10 mg of baicalein, were filled in hard gelatin capsules and placed in the dissolution vessel containing 900 mL of 0.5% SDS with phosphate buffer (pH 2.0 or 6.8). The vessel was maintained at 37 °C ± 0.5 °C and stirred at 100 rpm. Approximately 2.0 mL of sample was withdrawn from the dissolution medium and replaced by an equivalent volume of fresh medium at 5, 10, 20, 30, 45, and 60 min. The baicalein content was determined using HPLC after the sample was filtered through a 0.45 μm membrane. Experiments for each sample were performed in triplicate.A total of 24 male SD rats (SPF grade) weighing 220 ± 10 g were purchased from Benbu Yinuogui Biology Technique Ltd. The rats were kept in the Animal Research Center to acclimatize to a new environment. The rats were then randomly divided into four groups—namely, baicalein, BPC, TCS, and PM of TCS.All animal experimental protocols were approved by the Animal Care Committee of the Institute of Chinese Medicine in Jiangsu Province. The rats were fasted for 12 h and provided free access to water prior to the experiment. Each group was orally administered with a single dose equivalent to 40 mg/kg baicalein . An aliq2PO4 were added and vortexed for another 3 min. The mixture was centrifuged at 15,000 rpm for 5 min prior to evaporation under nitrogen. The dried residue was dissolved in 100 μL of methanol–distilled water . After being vortexed at 15,000 rpm for 5 min, the supernatant was injected for HPLC-ESI-MS/MS analysis. The MS/MS system  was operated under positive mode and multiple reaction monitoring mode. The MS conditions were as follows: ion spray voltage of +5.5 kV; nitrogen as nebulizer gas, auxiliary gas, and curtain gas at 30, 60, and 10 psi, respectively; collision gas set at medium; and auxiliary gas temperature of 400 °C.Baicalein, baicalin (corresponding to 5.75 and 5.06 μg/mL), 100 μL of plasma, 100 μL of mixed standard solution, and 10 μL of internal standard solution (5.60 μg/mL) were mixed and vortexed for 15 s. Approximately 0.5 mL of methanol and 100 μL of 1 M KHmax) and maximum time (Tmax) were directly determined from the concentration–time data. Other pharmaceutical parameters (AUC0–24 h and t1/2) were analyzed using pharmacokinetic program DAS 2.1.1. ANOVA was performed using SPSS 16.0 software.The maximum concentration  in distilled water was higher than that of BPC (5.02 ± 0.09 μg/mL) (p < 0.01). The solubility of TCS (230 ± 8.78 μg/mL) in n-octanol was slightly lower than that of BPC (260 ± 7.52 μg/mL). Moreover, TCS had lower log p values than BPC (2.01 vs. 2.04). The increase in solubility in distilled water and decrease in n-octanol may be caused by the natural hydrophilic structure of Soluplus. Therefore, TCS could enhance the water solubility of BPC.p < 0.01) compared with BPC, which peaked at 1.01 h (6.05 μg/mL). TCS exhibited a marked enhancement compared with BPC in oral bioavailability, with an increase in AUC0–24 h (53.16 μg·h/mL vs. 38.40 μg·h/mL) (p < 0.05), and AUC0–∞ (62.47 μg·h/mL vs. 50.48 μg·h/mL) (p < 0.05). The relative bioavailability of TCS was approximately 123.75% compared with BPC, confirming the enhanced bioavailability in the complex. Similarly, a four-fold increase in Cmax (25.55 μg/mL vs. 6.05 μg/mL) was observed.Plasma concentration–time profiles are presented in max and AUC0–∞ of BPC. All these results demonstrated that TCS may be applied to baicalein’s oral solid preparation.Soluplus was adopted as a hydrophilic pharmaceutical excipient to improve solubility, in vitro dissolution, or in vivo bioavailability in previous studies, such as solid dispersion , nanosusmax and AUC0–24 h, compared with BPC. The preparation method is simple and convenient, but also for Soluplus as a safe and effective drug excipient to explore a new pharmaceutical application. In conclusion, TCS is a promising method to improve the flowability and dissolution for drug–phospholipid complex.In our study, a novel TCS composed of baicalein, phospholipids, and Soluplus was successfully developed. The 35° angle of repose of TCS indicated an improvement in flowability, which met the industrial demand (θ < 40°). Moreover, TCS exhibited a marked enhancement in both the rate and extent of dissolution in vitro, as well as the bioavailability parameters C


To evaluate the effectiveness of hyperbaric oxygen in the treatment of patients with idiopathic sudden sensorineural hearing loss (ISSHL).An Embase, MEDLINE and Cochrane search were utilised to identify various clinical trials on the treatment of ISSHL. Studies that were published between 2002 and 2018 and written in the English, Dutch or German language were included. Search terms included synonyms for idiopathic sudden hearing loss.p = 0.005) of HBO + SS therapy on patients with an initial hearing loss of ≥ 81 dB.A total of 16 articles were identified regarding hyperbaric oxygen therapy. All patients were evaluated with pure-tone audiometry. A major part of the cases presented with unilateral hearing loss. In several studies, the average of the mean hearing gain at five contiguous frequencies was significantly higher in the hyperbaric oxygen (HBO) therapy and systemic steroid (SS) group in patients with severe or profound hearing impairment. They recorded a significant treatment effect (On the whole group of ISSHL patients, no significant difference was demonstrated between the intervention and control group. However, in severe or profound hearing-impaired ISSHL patients, significant benefit was observed in the intervention group. These results likely indicate that adding HBO to steroid therapies might be of benefit in cases of severe and profound hearing impairment. Idiopathic sudden sensorineural hearing loss (ISSHL) may be outlined as hearing impairment over at least 30 dB on three adjacent frequencies. It develops within an interval of maximum 3 days . The incThe prognosis of ISSHL is difficult to determine, if we take into consideration the possibility of spontaneous recovery. This phenomenon seems to occur in 29–78% of patients affected by ISSHL. The shape of the audiogram at onset of the disease may be a predictor of hearing recovery .In clinical practice, systemic steroid therapy (SS) has been the mainstay for ISSHL. More recently, intratympanic steroid (ITS) treatment has emerged and became more popular with otolaryngologists. This is an interesting option due to absence of unfavourable side effects  which are known in the systemic steroid treatment .Hyperbaric oxygen therapy has been commonly used for a long period of time. It was first used in the late 1970s by Goto and Vincey in the treatment of ISSHL , 7. GotoIn theory, a combination of treatments might provide a better outcome for patients with ISSHL. Therefore, this systematic review will investigate evidence for a possible effect of HBO treatment on hearing recovery in ISSHL patients.No ethical committee approval was required for this literature review. The systematic review was conducted according to the preferred reporting items for systematic reviews and meta-analyses (PRISMA) statement , 10.An electronic search in the Cochrane library, PubMed and EMBASE databases was performed on 19-05-2018. These searches were conducted to identify clinical trials on the treatment of idiopathic sudden sensorineural hearing loss. The PICOS structure was utilised to define the research strategy. It was mandatory for the published studies to be written in the English, Dutch or German language and published between 2002 and 2018. The following search terms were applied: sudden sensorineural hearing loss, sudden deafness and idiopathic sudden hearing loss. Restrictive search terms were hyperbaric therapy, oxygen therapy, corticosteroids and dexamethasone. The detailed search strings can be found in Appendix Each study had to be a clinical trial that compared the treatment of sudden sensorineural hearing loss with a control group. Intravenous, oral and intratympanic corticosteroid treatment comparing combinations with or without hyperbaric oxygen therapy, were included. Patients with a sensorineural hearing loss equivalent to at least 30 dB met the inclusion criteria.Several criteria for exclusion were applied: (1) patients who had another cause for their sudden sensorineural hearing loss such as noise, infections or ototoxic medication, (2) conductive hearing impairment, (3) case reports, and comments on articles and reviews, (4) Other languages than English, Dutch or German, (5) patients receiving therapy after 30 days of onset of ISSHL.After screening of title, abstract and full text, the remaining articles were critically appraised for risk of bias. Quality assessment of eligible studies was assessed according to the risk of bias assessment tool for non-randomized studies (RoBANS) .Study characteristics and outcome data of included studies were extracted, namely: publication year, study design, participants, type of treatment , duration and dosage of treatment, follow-up period and pure-tone average (PTA). The primary outcome was the PTA. The committee of the AAO-NHS, established various guidelines for diagnostic criteria in 1995 . To compAll groups are assessed based upon their hearing level. This hearing impairment has been categorised according to a standard which has been identified by the World Health Organisation (WHO) . No hearInformed consent was obtained from all patients. The participants were given oral information regarding the procedures by their ENT doctors.Figure According to the RoBANS, the risk of bias of the articles was independently appraised by two reviewers. All eligible articles had satisfactory quality therefore were included for data extraction. Table The total number of operated ears was 1759 of whom 580 were treated with HBO Table . Three dp = 0.014 and p = 0.005) [p < 0.05).Ten suitable trials involving 1295 patients were retrieved Table  17–25].–25.17–25= 0.005) . The sam= 0.005) . Patientp < 0.355) [p = 0.012) A different trial, had the same conclusion with regard to the significance of HBO therapy on patients with severe/profound hearing loss  . However55 . Howep = 0.754) [Three studies, conluded that the addition of HBO did not have a significant impact on the prognosis –21. Sata= 0.754) . In thre= 0.754) –25.p = 0.01). Recovery for patients suffering from profound and severe hearing loss was significantly associated with a therapy form based upon HBO + IVS (p = 0.046).Pezzoli et al., assessed 44 patients with ISSHL in their clinical trial . Mean PTp < 0.05) [In Pezzoli’s study, having experienced a moderate form of hearing impairment and increasing days after onset were negatively correlated with recovery. In Alimoglu et al., subjects whom received combination therapy in the first 15 days after onset, showed a statistically significant difference between mean gains ( < 0.05) .Ajduk et al. proposed that patients with an initial hearing loss of ≤ 60 dB had significant improvement at 500 Hz  Whereas p = 0.0014).Khater et al., treated 22 patients who suffered from ISSHL . The patp > 0.05).A clinical trial conducted by Chao et al. compared the usage of ITS in combination with HBO therapy . The heaIn this systematic review, we executed a search of the literature to identify evidence evaluating the effectiveness of hyperbaric oxygen therapy in ISSHL. Our search identified a total of 16 articles –30. In ap = 0.010). The same negative correlation has been recognised by various other reports [Various outcomes regarding the effectiveness of HBO are reported: eight out of sixteen articles, concluded that there was a significant difference between the intervention and control group –27, 29.  reports –38.Though early application of HBOT appears to be a logical advice, practical burden arises. Are there HBO therapy centers available in the region and are these capable of accepting this ‘new patient flow’ for a new indication (hearing recovery after ISSHL). And if so, is it cost effective? There is a considerable percentage of patients known to recover spontaneously . This rep = 0.014 and p = 0.005,respectively) [There is a significant higher impact of the combination therapy (SS + HBO) on ISSHL patients diagnosed with severe or profound hearing loss. Various studies have shown the hearing recovery in this specific category of patients , 23–30. ctively) . These rLamm et al., suggested HBO therapy as an adjuvant form of treatment after failure of conventional therapy in ISSHL . NonetheHBO is recognised as a safe treatment modality if the pressure is kept lower than three atmospheres (ATM) with sessions lasting up to 2 h . A studyHBOT was given to patients with ISSHL next to steroid medication . The variation in steroid regimen, method of administration and therapy duration differed distinctively among the studies. Also, the patients were not pooled correctly in some cases. These distinctions could potentially influence the comparability of various study cases and cause confounding bias. Though little is known about the final treatment outcome between the various application methods, it is highly unlikely the application method will interfere with hearing outcome . Most ofIn general, there was no significant difference observed between the intervention and control group. However, in severe to profound hearing loss, according to some reports, the patients might benefit from the adjunction of HBO to corticotherapy. Future prospective research, with larger series is required to further illuminate this topic.


Hearing impairment is a leading cause of disability globally and is particularly prevalent in elderly populations. Hearing aids are commonly recommended to mitigate the adverse effects on communication associated with hearing loss. However, the acceptability of hearing aids to elderly individuals is low and the majority of potential users do not wear hearing aids. Most hearing aids are designed with a discreet form factor in mind, to minimize device visibility. Given the range of comorbidities associated with hearing loss in the elderly, this conventional form factor may not always be optimal. The present study examined the experiences of elderly individuals with a recently developed, unconventional, body-worn hearing instrument, the EasyHear™ Grand . The bilaterally fitted instrument incorporates large controls, a color display, beamforming sound processing, and Bluetooth capabilities. Forty-three elderly participants  were surveyed to gauge level of benefit and satisfaction with the device and opinions regarding the hearing aid. They were assessed using three standardized questionnaires  and through open-ended, structured interviews. Participants rated their EasyHear device fitting highly for hours of use and improved quality of life and rated the device favorably for improved communication and benefit in background noise. A majority of users felt the device improved listening ability in their expressed area of greatest need, and also for their second highest prioritized area of greatest need. Less than 10% of users felt their listening was only occasionally or hardly ever improved when using the body-worn device. Benefit and satisfaction ratings with the EasyHear Grand were comparable to those in studies involving conventional form factor devices. Interviews highlighted areas where users felt the device could be improved—extra noise reduction, changes to device dimensions, receiver/eartip fit, and cableless technology were among the areas mentioned. Many participants valued smartphone linkage and Bluetooth capability. The EasyHear Grand, with its body-worn design and large, simple controls, was well accepted by the majority of participants. Hearing aids that break from conventional design formats may benefit many elderly individuals with hearing impairment and promote increased user acceptability. Hearing loss is one of the principal global causes of disability  and the As yet, there is no pharmacological intervention for presbycusis and the most commonly advised treatment is use of an amplification device. This typically involves unilateral or bilateral hearing aid fitting. A wide range of amplification devices are available for individuals with presbycusis and over 15 million hearing aids are globally fitted each year , primariPresent-day hearing aids have a limited range of form factors. The vast majority of hearing aids are based on behind-the-ear (BTE) or in-the-ear (ITE) designs. These designs may not be appropriate for all elderly individuals with hearing loss. Many elderly persons with hearing impairment have comorbidities such as impaired vision, limited sensation of touch, limited range of movement, and dementia , 15–18. For potential elderly amplification device users with such issues a larger instrument that is easier to see and manipulate has benefits . The forA recently developed hearing aid, the EasyHear™ Grand , focuses on assisting persons with hearing impairment to combat real-world noise and enhance mid-range, face-to-face communication. The hearing aid was designed for a variety of users, but particularly for elderly clients who seek an easy to use and robust hearing device. The EasyHear Grand is a body-worn hearing instrument  providing unilateral or dual, independent, right/left channel bilateral amplification. The hearing aid uses a 3.8V Li-ion rechargeable battery with a typical battery life of 10 hours and a 6-6.5 hour charging time. The EasyHear Grand has a frequency range of 300–8000 Hz with a maximum power output of 136 dB SPL at 900 Hz. The EasyHear device allows wearers to use a proprietary beamforming technology  that focAnecdotal reports from EasyHear clients suggested that the device improved listening ability for many of its users. However, no formal clinical studies had been conducted to evaluate the device with a senior client group. Indeed, in general, the effectiveness of nonconventional hearing aids is rarely evaluated .One possible way to improve hearing aid acceptance is to collect information from the user perspective in the fitting trial period, particularly after user experience in everyday listening environments, and use this information to reflect upon future device adaptations and enhancements. This strategy has been considered essential since the early days of hearing aid provision , 30. SelUser benefit and satisfaction with the EasyHear Grand were investigated in the current study. Self-reported outcome measures—questionnaires and open-ended interviews—were completed before and after fitting to evaluate how effective the device may be with elderly users in everyday listening situations. Standardized questionnaires and open-ended interviews were employed to provide (a) ratings of benefit and satisfaction with the EasyHear Grand device and (b) an understanding of the elderly client experience with this type of hearing aid. The study aimed to consider this device in relation to the globally increasing need for effective solutions to presbycusis-related hearing loss.The present study examined user benefit and satisfaction with the EasyHear Grand for individuals who had a bilateral hearing impairment, with moderate to severe mixed or sensorineural hearing loss in the better ear, and with or without previous experience with hearing amplification devices. The prospective, repeated-measures study was carried out over eight weeks with each participant. A set of standard, self-reported outcome measure questionnaires were used as well as an open-ended interview session to collect information regarding user benefit and satisfaction ratings with the EasyHear Grand. An eight-week final review point was chosen because outcomes from hearing aid fitting are stable after approximately six weeks post-fit .A new EasyHear hearing device was provided to Cantonese-speaking persons with moderate to severe bilateral sensorineural hearing loss. Participants were recruited through EasyHear clinics at two localities in Hong Kong, China. Participants who agreed to join the program and who are within the inclusion criteria underwent diagnostic audiological assessment and cognitive screening assessment. They were prescribed with the device by a clinical audiologist based at the hearing clinics. Study participants were recruited (a) during a routine EasyHear Grand hearing aid first fitting session, or (b) by offer of a free trial with the EasyHear Grand. Participants were verbally informed about the aims of the study and written information was also provided. All participants visited their hearing clinic three times during the study, including a long-term follow-up visit eight weeks after initial EasyHear fitting. Assessment duration was approximately 60 minutes per visit. Written consent was obtained from each participant and the research protocol was approved by the Human Research Ethics Committee, The University of Hong Kong, prior to study commencement. A HKD 200 payment was made to subjects who completed the three visits, as compensation for travel costs.Formal inclusion criteria were (1) above 40 years of age at commencement of the study; (2) Cantonese as the primary or secondary spoken language; (3) mixed or sensorineural hearing loss with the pure tone average at 0.5, 1, and 2 kHz in the better hearing ear > 40 dBHL and ≤ 90 dBHL; (4) both first time users and experienced hearing aid users; (5) adequate physical and cognitive status to enable the individual to follow the study protocol; and (6) willing to participate and to comply with all requirements of the protocol. Formal exclusion criteria were (1) cognitively impaired; (2) unwilling or unable to complete all testing sessions; (3) asymmetrical hearing loss with a difference of pure tone average between the left and the right ear of more than 30 dBHL; (4) purely conductive hearing loss in one or both ears; and (5) previous user of an EasyHear device.Initial collection of participant demographic details, and medical and hearing health history, was followed by recording of pure tone audiometry results from assessment at the clinic. Then baseline data were compiled using the following assessments: Montreal Cognitive Assessment (MoCA) test (Hong Kong version); International Outcome Inventory for Hearing Aids (IOI-HA) questionnaire; Chinese Profile of Hearing Aid Benefit (C-PHAB) questionnaire; and the Client Oriented Scale of Improvement COSI questionnaire. Visit 1 was split into 2 parts, with the first part (Visit 1a) focused on collection of participant demographic details, medical and hearing health history, audiometric and cognitive (MoCA) assessment, and hearing aid fitting, and the second part (Visit 1b), two to seven days later, focused on completing the COSI, IOI-HA, and C-PHAP questionnaires. The IOI-HA, C-PHAB, and COSI questionnaires were administered again at the second and final visits. At the final visit participants were also asked a series of open-ended interview questions about their experience with the EasyHear Grand. All responses were encouraged, whether they were positive or negative about the device. The Montreal Cognitive Assessment (MoCA) test is a 30-item questionnaire used to assess mild cognitive impairment , 33. TheThe EasyHear Grand is an air conduction amplification device intended to be fitted in a hearing health care setting and not purchased as an over-the-counter device. The fittings were all bilateral, with maximum output and frequency response characteristics prescribed for each individual ear using the manufacturer's proprietary fitting algorithm. Participants were mostly fitted with standard, closed-dome earmolds. If a closed-dome earmold did not fit well, or if acoustic feedback occurred, custom earmolds were manufactured for the participants. The elderly participants were allowed to freely control the device, including volume and listening mode selection (noise-cancelling plus beamforming or noise-cancelling only modes) during the fitting period. The hearing aid is typically placed in a shirt pocket or hung from a lanyard in front of the chest. A single, omnidirectional microphone is located on both the left and right earphone cables of the EasyHear Grand . The earThe test device was fitted at Visit 1 according to each participant's most recent pure tone audiometry results. The audiogram was entered into a proprietary desktop computer program. The software generated the initial fitting parameters . These custom settings were then entered into the EasyHear Grand. Fitting parameters were ear specific for this device as bilateral fittings leading to aided binaural hearing were standard. The hearing aids were fine-tuned at later visits if a feedback problem or discomfort from the amplified sound was noted. The test devices were fitted with one predefined listening program for the test period. To reduce the potential for bias effects, data at Visits 2 and 3 were obtained by university research assistants who were not affiliated with the EasyHear clinics and were known by the participants to be unaffiliated to the clinic.Both qualitative and quantitative analyses were undertaken. Quantitative statistical analyses for questionnaire test scores at Visit 1, Visit 2, and Visit 3 were carried out, along with qualitative thematic analysis of the open-ended interview responses obtained at Visit 3. A significance level of p<.05 was set for all quantitative analyses.All participants passed the MoCA evaluation . Nine potential participants were excluded from the study because they did not achieve a MoCA score of ≥ 26 points. Eight additional potential participants dropped out of the study before completion because they had left Hong Kong (n=1), could not comply with the revisit schedule (n=5), or returned the device because they feared losing it before the end of the study (n=2).For each of the seven IOI-HA questions a score of five indicates the best possible outcome and a score of one indicates the worst. Domains are (1) use of the hearing aid, (2) benefit from the device, (3) residual difficulties when wearing the hearing aid, (4) overall satisfaction with the device, (5) residual participation restriction (how much does hearing loss still affect daily life), (6) impact on others, and (7) quality of life change. The mean overall IOI-HA scores were 3.59, 3.83, and 3.86 for Visits 1, 2, and 3, respectively. Mean scores for each of the seven question items and the overall score are shown in Repeated measures ANOVAs were carried out for all four C-PHAB domains over the three visits with the participants wearing their EasyHear devices. Significant differences were found for ease of communication benefit =5.2, p=.01), background noise benefit =3.49, p=.04), and aversiveness benefit =5.17, p=.02) across visits. The Reverberation questions were completely answered by only nine of the participants see . Many ofWhen asked to identify the main areas where they needed assistance from a hearing aid all participants (n=43) nominated at least one specific need, and virtually all could nominate a second need (42 participants). Over half the participants could nominate a third specific need (n=25) but only two participants nominated four specific areas they needed help with.For specific need 1 the most common priorities were face-to-face communication, listening at a distance from a speaker, and telephone conversations. For specific needs 2 and 3, listening to speech in noise and watching television were often additional priorities. The range of needs expressed by participants at their first visit is summarized in The COSI questionnaire also allows participants to rate the final degree of improvement they feel their hearing aid has provided—from “hardly ever” any improvement (10% improvement only) to “almost always” improvement (95% improvement). The COSI results for the degree of improvement for main expressed needs and for specific needs 1, 2, and 3 at Visits 2 and 3 are summarized in At the final visit each participant was asked a series of interview questions about their experience with the EasyHear Grand. A summary of responses to the six questions asked is shown in Participants commented on both the hardware and software features of the device and the most common suggestions for possible improvements are noted in IOI-HA scores were evaluated to gauge the EasyHear Grand wearer experience. On average, participants used their hearing aid between 4 and 8 hours each day. Participants still had difficulty in the communication situation they most wanted improved hearing, but this difficulty was now only slight on average and participants generally now felt their hearing loss only slightly affected their everyday activities and that their family and friends were now only slightly bothered by their hearing loss. EasyHear Grand users felt that, overall, their device had made “quite a lot” of improvement to their enjoyment of life.As the IOI-HA, PHAB, and COSI have been previously used, it was possible to compare EasyHear Grand group results with other studies. In the only directly comparable Hong Kong study  the meanParticipants in less recent IOI-HA studies wore analog or early digital technology devices. It has been suggested that scores using more advanced devices may be higher . TherefoMean IOI-HA scores showed slight but statistically significant improvement over time. Generally, only small changes in IOI-HA scores occurred between visits although a trend can be seen (in six out of seven questions) for scores to improve between initial fitting and final visit. This suggests that the EasyHear Grand users noted positive changes soon after they first experienced their changed auditory environment and they did not alter their opinions very much over the following months of hearing aid use.Three C-PHAB question areas were fully answered over all visits and significant improvements were noted in participant responses over the three visits for these areas—ease of communication benefit, background noise benefit, and aversiveness benefit. The reverberation area had low response numbers and therefore this area and the global score were not included in the final analysis. A previous Hong Kong study  found C-The specific needs mentioned by EasyHear wearers in the COSI questionnaire were typical of those expressed by most hearing clinic clients. The common priorities were face-to-face communications, listening at a distance from a speaker and telephone conversations, along with listening to speech in noise and watching television. For a somewhat younger group of clients, with milder hearing loss, the previous Hong Kong study  found coA substantial majority of EasyHear study participants felt the device improved their general hearing ability and made speech clearer—95% of users felt this was the case. Many participants also considered that the EasyHear Grand improved their ability to listen in noise (33%). Noise reduction was highlighted by participants as being a specific benefit of the hearing aid. Very few (only 5%) considered that the device made no difference to audibility or made listening a worse experience. Future research work to quantify expected improvements in speech perception using the EasyHear Grand beamforming technology would be useful. Speech perception studies with the EasyHear Grand in controlled quiet and noisy environments would provide a better understanding of the effects of the amplification system on elderly listeners with hearing loss.Around a quarter of participants (n=11) mentioned Bluetooth functionality as a specific positive feature of the EasyHear Grand. This suggests that many of the elderly participants in this study were making use of this function to link with their smartphone or other devices. However, a number of participants also mentioned difficulty using the Bluetooth function and this suggests there is room for further device improvement or client education in this area.Noise is a perennial problem for hearing aid users. The EasyHear Grand uses beamforming and other technology to reduce the impact of background noise on speech but—like all hearing devices—cannot eliminate noise entirely. Although many clients praised the EasyHear Grand's success in reducing background noise problems, noise was still a problem for listeners. The most frequent suggestion for improvement was for further “noise reduction.” Additionally, some participants felt that device size could be reduced and that there were problems with earphone stability and/or comfort in the ear. Further thought to earphone and earmold design may be useful. The increasing use of wireless technology in many electronic devices may have influenced the large number of respondents (n=28) who advised removing wires from future EasyHear models. Obviously a wireless instrument increases convenience for wearers but potential disadvantages may be involved, such as increased power consumption and device size. Several participants suggested adding a clip on the rear face of the EasyHear Grand for easier attachment to clothes.Previous research considered the reasons that elderly Chinese people with hearing loss in Hong Kong do not seek out hearing aids . AffordaFor this study only EasyHear Grand users without cognitive impairment were recruited. The effectiveness of the EasyHear Grand with cognitively challenged users is unknown. The study used standardized questionnaires that are widely used in rating client benefits and satisfaction with hearing aids. However, some of the questions were inappropriate for participants in this study (such as the C-PHAB reverberation items) and this limited the dataset obtained in the study. The open-ended interview used a broad but restricted question set—a less structured interview may have gained more insights into client attitudes and feelings about the EasyHear Grand. Client outcomes for the EasyHear Grand fittings were assessed within an eight-week timeframe. A longer term study—for example, over a 12-month period—may better map client outcomes postfitting. The majority of participants were elderly persons who may require longer fitting periods to fully adapt to hearing aid use.A main feature of the EasyHear Grand device is its beamforming microphone function. However, the present research study was not designed to assess beamforming capabilities. Beamforming technology has potential advantages over conventional microphone designs to reduce competing noise and enhance speech in everyday listening situations. Research comparing speech recognition scores in an elderly client group for different microphone, noise, and environment parameters could quantify the effectiveness of this type of device technology.Overall, the study participants expressed clear satisfaction with their EasyHear Grand device. Average ratings with the EasyHear Grand were equivalent to those achieved in other professional hearing aid fittings. For example, participants rated their EasyHear device fitting highly for hours of use, perceived benefit, satisfaction with the fitting outcome, and improved quality of life using the International Orientation Inventory-Hearing Aid questionnaire. Similarly, wearers rated the EasyHear positively for improved communication and benefit in background noise with the Profile of Hearing Aid Benefit questionnaire. The Client Oriented Scale of Improvement results showed that a large majority of EasyHear Grand users (81%) felt the device had greatly improved their listening ability in their main situation of need and also for their second listening priority (72%). Less than 10% of users felt their listening was only occasionally or hardly ever improved when using the EasyHear Grand. Participants gave a wide range of comments during their open-ended interview sessions. Nearly all the participants (41 out of 43) felt the device improved their listening ability. Nearly half the study participants had prior hearing aid experience and virtually all of this group considered that the EasyHear had improved sound quality compared to their previous hearing aid (20 out of 22 participants). The noise reduction performance of the EasyHear was valued by many participants. Interviews also highlighted areas where users felt the device could be improved—extra noise reduction, reduced device size, earphone/earmold design, wireless technology, and a pocket clip were among the items mentioned. Many of the senior clients in this study were “technology aware”—interview comments indicated that many of the elderly clients valued bilateral smartphone linkage and Bluetooth capability. Few participants expressed negative comments directly related to the body-worn form factor of the hearing aid , and none expressed a desire to wear an alternative BTE/ITE design.The study findings suggest that the EasyHear Grand, with its body-worn format and simple controls, provides a level of benefit and satisfaction that is comparable to that reported for persons with hearing loss professionally fitted with a range of conventional design form hearing aids. The majority of elderly users in the present study expressed positive outcomes with the hearing aid fitting. Conventional hearing aid design formats may not be optimal for all elderly individuals with hearing impairment and innovative designs—such as those used in the EasyHear Grand—deserve consideration. An increased range of hearing aid design options may raise the currently low level of hearing aid usage in elderly populations.


Human apolipoprotein B mRNA-editing catalytic polypeptide-like 3 proteins (APOBEC3s or A3s) are cytosine deaminases that protect cells by introducing promutagenic uraciles in invading retro-elements. However as a drawback of this protective activity, A3s can also target cellular DNA, leading to DNA damage and to the accumulation of somatic mutations that may contribute to tumorigenesis. Among A3s, A3A has been shown to be particularly proficient at mutagenizing cellular DNA, but whether this enzyme exerts additional effects on the cellular physiology remains unclear. Here, we show that A3A editing of cellular DNA leads to reactive oxygen species (ROS) production through Nox-enzymes. ROS production occurs in two distinct model cell lines and it is contingent upon DNA replication and intact enzymatic properties of A3A. For the first time, our results indicate that the editing activity of A3A results in the induction of a pro-inflammatory state that may possibly contribute to the constitution of a tumorigenic-prone environment. A3s use preferentially single-stranded DNA as substrate of their enzymatic activity and catalyze the deamination of cytosines into uracils6. Cytosine deamination does occur spontaneously in cellular DNA, but in this case uracils accumulate at a much lower rate and are quickly disposed of by dedicated cellular enzymes8. In the case of invading retro-elements, A3s introduce a large number of mutations on the negative strand DNA that is then used as a template for the synthesis of the positive strand one during reverse transcription5. As a result, mutations become fixed on the viral genome as G to A transitions, ultimately leading to the element inactivation by mutagenesis14. In addition to this mechanism of inhibition, A3s has been also described to act through alternative mechanisms. Indeed, A3G is able to directly interfere with the process of reverse transcription through a cytosine-independent mechanism in the case of HIV-117 and appears to inhibit indirectly Measles virus replication by modulating the activity of the mammalian target of rapamycin complex-1 (mTORC1)18.Apolipoprotein B mRNA editing catalytic polypeptide-like 3 proteins  are a family of cytosine deaminases composed of seven distinct members in humans (named A to H)non-self genetic elements, A3s expression may lead to the accumulation of somatic mutations27. These observations are of importance given that cancer genomic studies are unveiling the presence of an higher than expected accumulation of G to A transitions in nucleotide contexts evocative of A3s in cancer cells37. While these observations leave open the question of causality between editing and tumorigenesis, they clearly raise the possibility that cytosine deaminase enzymes may be involved either directly or indirectly in this process.A growing number of studies are revealing that as a drawback of what is a protective role of the cellular genome from invasion of 38, but also cellular DNA39. Expression of A3A induces a strong activation of several key mediators of the DNA damage response pathway, as the phosphorylation on Ser139 of the histone variant H2AX, the recruitment of 53BP1 and of the Replication Protein A (RPA) proteins and ectopic expression of A3A leads to cell cycle arrest and cell death39. Several studies have firmly linked these effects to the direct deamination of the cellular genome by A3A through its transient access to single-stranded DNA intermediates during cellular DNA replication26, followed by the action of Uracil-DNA glycosylases (UNG) and the recruitment of the apurinic/apyrimidinic (AP) endonuclease that create a site of lesion on the host genome. To add to the complexity of its action in cells, A3A appears regulated through multiple layers of control among which its nucleocytoplasmic distribution, or its interaction with cellular cofactors that influence its stability and enzymatic activity42.Among the members of the A3 family, A3A has received an increasing attention as a nuclear enzyme endowed with a proficient ability to deaminate not only foreign DNA introduced within the cell by transient transfection44. We further determine that ROS production depends on the catalytic activity of A3A and that it is observed upon expression of both described A3A isoforms. These findings strongly support a previously proposed model45 in which contrarily to the well-described property of ROS to induce DNA damage, DNA damage may also initiate ROS production.In this work, we have used the controlled expression of A3A in two model cell lines  to explore the possible consequences of the expression of A3A in different cellular contexts. For the first time, we show here that the DNA damage induced by A3A leads to the production of reactive oxygen species (ROS) produced by NAD(P)H oxidases (or Noxes)27 and suggest that both DNA damage and ROS production are unlikely to play a role in the antiviral properties of A3A, at least as it can be appreciated in non-dividing DCs. Rather, these findings suggest that these effects are a deleterious consequence of the mode of action of A3A in proliferating cells in which A3A not only directly mutagenizes the cellular genome, but also indirectly contributes to promote a proinflammatory, potentially pro-tumorigenic environment via ROS.Given that ROS are well described inducers of DNA damage, we explored the possibility that they could exacerbate the extent of DNA damage already induced by A3A. Through the use of Nox inhibitors, we show that this is not the case, indicating either that the levels of ROS produced in this context is not sufficient to induce DNA damage, or that their effects is masked by the massive action of A3A. Contrarily to what observed in replicating cells, DNA damage as well as ROS production are not observed upon A3A induction in differentiated U937 cells, nor in dendritic cells (DCs) differentiated from primary monocytes and further stimulated with interferon alpha (IFNα), a strong inducer of A3A expression. Thus, these findings are in agreement with previous studies suggesting a strong requirement for cell cycle progression for the effects of A3AOverall, our study contributes to the understanding of the properties of A3A by highlighting for the first time that through a DNA damage response, A3A is likely to specify a more complex cellular program than previously anticipated.45). ROS play numerous roles in the cell physiology beyond the induction of cell death, under both normal and pathological conditions46 and their production in the context of the A3A-induced DNA damage would offer a different perspective on A3A.A3A induces massive editing of cellular DNA, resulting in the induction of a strong DNA damage response that leads to cell death. While solid experimental evidence supports these effects, the physiological consequences of the expression of A3A beyond DNA damage and cell death remain unclear. As schematically presented in Fig. 39, and U937 cells, cells of myeloid origins that can be induced to differentiate into a non-dividing macrophage-like status upon PMA treatment47. To compare the expression levels of A3A in our dox-inducible cell lines with the ones observed in primary cells we used monocyte-derived dendritic cells  and the antibody ApoC1748. This antibody is a particularly useful tool as despite the fact that it was originally raised against A3G, it also recognizes A3A but not the other members of the A3 family . Given that A3A and A3G bear distinct molecular weights , this antibody allows an easy discrimination between these two proteins upon SDS-PAGE gel migration and WB. In our hands, U937 do not express detectable levels of A3A even after concomitant PHA and IFNα stimulation, -dependent promoter. We selected in particular two model cell lines: HeLa cells that have been widely used in the past to explore the functional consequences of A3A expressionA3A expression was induced for twenty-four hours prior to WB analysis in HeLa as well as in U937 cell lines either cycling or growth-arrested upon incubation with PMA that induces their differentiation into macrophage-like cells Fig. . Given t139, γH2AX, as described in25, Fig. 50 and validate the experimental cellular models used in our study.The extent of DNA damage was then measured by flow cytometry upon staining with an antibody specific for the phosphorylated form of the histone variant H2AX, a well described marker of DNA damage . The fluorescence of DCFDA relies on oxidation and therefore DCFDA staining is a widely used technique to measure the accumulation of reactive oxygen species.Under these conditions, a significant ROS production was observed in cycling HeLa and U937 cells Fig. .The production of ROS required the cytosine-deaminase activity of A3A, as no ROS were observed in cells expressing the catalytically-inactive A3A mutant (E72Q) and similarly, no ROS were produced in the absence of DNA damage in PMA-treated growth-arrested U937 cells.Overall, these results indicate for the first time that A3A leads to ROS production in two distinct model cell lines and that this effect relies on the ability of A3A to attack cellular DNA through its cytosine deamination activity.51, MEASPASGPRHL). Whether this shorter A3A isoform exhibits distinct behavior than the full length remains unknown and it was therefore important to determine if ROS production could be differentially mediated by one or the other isoform. To address this possibility, we engineered either the shorter A3A isoform  and the longer one , a well-described broad Nox inhibitor was used in cycling U937 cells in which A3A expression had been induced. Under these conditions, DPI treatment of A3A-expressing cells completely abolished ROS production . Members of this family share the presence of at least six trans-membrane helices, of a flavin adenine dinucleotide- and of a NAPDH-binding domain and display an heterogeneous pattern of tissular expression and of intracellular distributionThe results presented above support the notion that the activity of A3A is accompanied by the production of ROS. In turn, ROS are known to exert pleiotropic effects on the cell, among which the induction of DNA damage itself. To determine whether the ROS produced here upon A3A induction could contribute to -and perhaps amplify- the DNA damage response commonly attributed to A3A, a time curve analysis was performed in cycling U937 cells. To this end, cell aliquots were harvested at different times post A3A induction and the extent of DNA damage and ROS production were measured by FACS Fig. . Under tImportantly however, inhibition of Nox enzymes through the use of DPI failed to prevent the accumulation of γH2AX-positive cells indicating that, at least under the experimental conditions used here, ROS do not further contribute to the DNA damage induced by A3A.A3A expression is commonly associated to cell death in cycling cells and given that ROS are also cytotoxic we set out to determine whether the A3A-dependent ROS production could contribute to the cytotoxic effects ascribed to A3A. To this end, we incubated cells with or without the ROS buffering agent NAC, that efficiently counters ROS production in cells Fig. , and we The results presented above as well as data in the literature indicate that cell cycling is a determining factor in the susceptibility of a given cell to the DNA damage and ROS production mediated by A3A. On the other hand, we have previously determined that A3A contributes to the modulation of HIV infection in myeloid cells and more specifically DCs and macrophages. Given that these cells act as central players of immune responses and that ROS have been described to be an integral part of antiviral responses, it was important to determine whether the expression of A3A in primary DCs could lead to a DNA damage response and to ROS that could in turn contribute to the poor susceptibility of these cells to viral infection or that could more generally play a role in the antiviral functions of DCs.As expected, treatment of DCs with IFNα strongly stimulated the expression of A3A Fig. . HoweverOverall, these results indicate that cell cycle progression is a general determinant in the susceptibility of target cells to both DNA damage and ROS production induced by A3A and suggest that these effects are unrelated to the antiviral functions played by A3A, at least in myeloid cells.53. The strong requirement for cell cycle progression for the effects of A3A was also underlined by the fact that no DNA damage nor ROS were produced in either PMA-differentiated U937 cells, nor primary DCs, irrespectively of the manner in which A3A was induced . These results therefore indicate that the effects of A3A are heavily dependent on the cellular context in which this protein is expressed and the fact that myeloid cells  exhibit a non-cycling status is likely the main reason why these cells are protected from the deleterious effects of A3A.The major novelty of our study is to present evidence that the DNA damage response orchestrated by A3A culminates in the production of reactive oxygen species (ROS) through Nox enzymes, linking for the first time the mutagenic activity of A3A to a functional consequence outside the direct deamination of cellular DNA. This activity is observed in the two model cell lines tested and is dependent on active cell cycle progression, in line with the higher availability of single-stranded DNA substrates during DNA replication54. In one of them, several DNA-damaging compounds were shown to drive ROS production via a Rac1-dependent mechanism that converged in the activation of Nox enzymes45. In line with this proposed mechanism of reverse control of ROS by DNA damage, we also show that a different DNA-damage agent, A3A, leads through cytosine deamination to the production of ROS by activation of Nox enzymes. In our hands, the levels of ROS generated via A3A do not exacerbate the extent of DNA damage already caused by A3A, nor its ability to cause cell death, implying that the direct deamination of the cellular genome by A3A plays a dominant function on these phenotypes and suggesting that ROS may act as second messengers, or mediate additional effects of A3A that remain to be discovered.While the presence of reactive oxygen species in the cell is well known to cause DNA damage, the possibility that DNA damage itself can drive the production of ROS has been evoked only in few studies39 and could in this manner contribute to tumorigenesis in vivo.A number of well-conducted studies firmly established A3A as a potent mutagenic factor whose activity on genomic DNA results in extensive DNA damage in model cell lines65.In addition to a direct contribution to this process through the introduction of somatic mutations in the cellular genome, our results indicate that A3A may also contribute to create an environment favorable to tumorigenesis through the induction of ROS. Indeed, ROS intervene in numerous cellular processes and clearly participate in chronic inflammation and have been clearly involved in the establishment of a pro-inflammatory, tumor-prone environmentTo conclude, the results presented in this study indicate that A3A exerts far more complex effects on the cell physiology than previously appreciated and indicate that this enzyme bears the potential to favor the process of tumorigenesis through both direct and indirect mechanisms.wild-type and mutant A3As were inserted in a pRetroXtight plasmid background (Clontech) by standard molecular biology techniques . This vector DNA allows the generation of doxycycline-inducible, stably-transduced target cells upon retroviral-mediated gene delivery . Antibodies used for WB were as follows: anti-apoC17  after a Ficoll and then a Percoll gradient. The enriched monocyte fraction was then purified to homogeneity by negative depletion with a cocktail of antibodies-coated beads that removed contaminant cells . Cytokines were purchased from Eurobio . GM-CSF and IL4 were used at a final concentration of 100 ng/ml each for four days, while IFNα was used at 1.000 U/mL for twenty-four hours prior to analysis. Blood material consisted of discarded “leukopacks” obtained anonymously from the EFS-Lyon. Gender, race, and age of donors are unknown to the investigator as is the inclusion of women, minorities or children. This research is exempt from approval, although written informed consent was obtained from blood donors to allow use of their cells for research purposes.To induce macrophage-like differentiation, U937 were treated with phorbol-12, myristate-13, acetate  for 2 days. Primary monocyte-derived dendritic cells were differentiated from blood monocytes of healthy donors upon incubation with 100 ng/ml of both granulocyte-macrophage colony-stimulating factor and interleukin 4  along with two miniviral genomes both pRetroX-based . The first bears the A3A sequences under the control of a doxycycline-inducible promotor, the second codes for the rtTA transactivator . Virions released in the supernantant of transfected HEK293T cells were then purified through a 25% sucrose cushion, resuspended and their infection titers determined by exogenous-RT activity against standards of known infectivity. Cells were then transduced at a multiplicity of infection of 10 and stable cell lines were obtained upon selection with Puromycin and G418 present on the two different pRetroX constructs.Murine leukemia virus (MLV)-based retroviral vectors used here for gene transduction have been described beforeDNA damage was measured by flow cytometry with an Alexa Fluor 488-conjugated monoclonal antibody directed against the Ser139-phosphorylated form of the histone variant H2AX . Unless otherwise specified, dox-treated cells were analyzed 36 to 48 hours after A3A induction. Cells were washed then fixed and permeabilized  prior to incubation with the above-mentioned antibody. Etoposide was used as a positive control . The extent of cell death was measured by propidium iodide staining by flow cytometry .5 cells were incubated with 20 μM of the cell permeable fluorogenic dye 2′,7′–dichlorofluorescin diacetate (DCFDA) for 4 hours, prior to flow cytometry analysis. DCFDA is cleaved by cellular esterases within the cell, yielding a metabolized form that becomes fluorescent upon oxidation. The oxidative stress inducer, tertiary-butyl hydroperoxide (TBHP at 110 μM) was used as a positive control and for these analyses it was added 30 minutes after incubation with DCFDA for a total of 3.5 hours, prior to analysis. When specified the Nox inhibitor Diphenyleneiodonium  and the ROS buffer N-acetylcysteine  were added to the culture media at the time of induction of A3A.ROS production was performed using a cellular ROS detection assay kit, according to the manufacturer’s specifications . Briefly, A3A expression was induced by incubation with dox and cells were then analyzed 36 to 48 hours post induction. Four hours prior to analysis, cells were counted and 10In all cases, cells were analyzed on a FACSCalibur (BD Biosciences) using CellQuest Pro (BD Biosciences).Student t tests were performed to determine statistically significance between the indicated conditions in the different experiments performed in this study, as specified in each figure legend.Supp Figure 1, original blots


Large mesopores of chiral silica nanoparticles applied as drug carrier are worth studying. In this study, chiral mesoporous silica nanoparticles (CMSN) and enlarged chiral mesoporous silica nanoparticles (E-CMSN) with a particle size from 200 to 300 nm were synthesized. Fourier transform infrared spectrometer (FTIR), circular dichroism spectrum, scanning electron microscopy (SEM), transmission electron microscope (TEM), and nitrogen adsorption/desorption measurement were adopted to explore their characteristics. The results showed that the surface area, pore volume, and pore diameter of E-CMSN were higher than those of CMSN due to enlarged mesopores. Poorly water-soluble drug nimesulide (NMS) was taken as the model drug and loaded into carriers using adsorption method. After NMS was loaded into CMSN and E-CMSN, most crystalline NMS converted to amorphous phase and E-CMSN was superior. The anti-inflammatory pharmacodynamics and in vivo pharmacokinetics results were consistent with the wetting property and in vitro drug dissolution results, verifying that NMS/E-CMSN exhibited superior NMS delivery system based on its higher oral relative bioavailability and anti-inflammatory effect because its enlarge mesopores contributed to load and release more amorphous NMS. The minor variations in the synthesis process contributed to optimize the chiral nano-silica drug delivery system. In recent years, mesoporous silica nanoparticles (MSN) fabricated based on various kinds of templates have aroused a lot of interest owing to their rigid frame, good biocompatibility and biodegradability, unique structural features , and facile surface modification ,2,3,4. TIt is widely accepted that CMSN can be applied as drug carrier to establish a profound of drug delivery systems, primarily covering controlled release, stimuli-response release and multifunctional release ,18,19. OThough great efforts have been made to study the synthesis process and application of CMSN with enlarged pore size, the potential ability of its drug delivery effects should be systematically elucidated ,26. In t−1 and 465.7 cm−1, Si-O-Si symmetric stretching vibration at 798.4 cm−1 and 792.9 cm−1, asymmetric stretching vibration absorption peak of Si-O-Si at 1080.9 cm−1 and 1075.6 cm−1, OH of Si-OH antisymmetric stretching vibration at 3425.2 cm−1 and 3425.5 cm−1, suggesting that the silica frame was successfully formed. Furthermore, N-H deformation vibration at 1635.2 cm−1 and carbonyl stretching band at 1699.2 cm−1 that shown in spectrum of CMSN originated from the chiral groups that grafted onto the silica frame [−1 and carbonyl stretching band at 1698.7 cm−1 from the spectrum of E-CMSN), implying that the existence state of functional groups in E-CMSN can slightly different from CMSN for the pore enlargement management. Circular dichroism spectrum is a critical parameter to confirm the chiral property. As shown in As shown in 2/g; E-CMSN: 474.360 cm2/g) and pore volume , revealing that the pore enlarging effect of E-CMSN is effective. Furthermore, pore diameter intuitively reflected the pore enlarging phenomenon since the pore diameters of CMSN and E-CMSN were 2.2 nm and 4.3 nm. The pore enlargement effect of E-CMSN was obvious since most common pore diameter of mesoporous silica nanoparticles was in the range of 2 to 3.5 nm [CMSN and E-CMSN morphology were studied using SEM and TEM, and the images were shown in o 3.5 nm ,29.2/g; decreased pore volume: 1.021 cm3/g) were higher than those from CMSN and NMS/CMSN , further confirming the superior drug loading capacity of E-CMSN.The drug loading capacity of NMS/CMSN and NMS/E-CMSN were 24.51% ± 0.27% and 27.45% ± 0.19% respectively. The loading capacity of NMS/E-CMSN was higher than that of NMS/CMSN even though their differences were not significant, illustrating that the enlarged pores of E-CMSN exhibited potential ability to entrap more NMS molecules. After drug loading, the drug crystalline state property was studied using BET, XRD, and DSC measurements. According to the BET result , all theXRD and DSC measurements explained the drug crystalline state after being loaded into the two carriers ,31. As sIn vitro dissolution of NMS, NMS/CMSN and NMS/E-CMSN in pH 6.8 PBS medium was shown in max and AUC. The relative bioavailability of NMS/CMSN and NMS/E-CMSN reached to 765.76% and 905.04%, respectively, thereby enhancing contributed the enhanced NMS dissolution based on the change of crystalline NMS to amorphous NMS using CMSN and E-CMSN. The result also confirmed that E-CMSN with enlarged mesopores was able to perform superior NMS delivery effect owing to its high oral bioavailability [The in vivo biological effect of NMS/CMSN and NMS/E-CMSN was studied by in vivo pharmacokinetics, anti-inflammatory pharmacodynamics, and mucous membrane adhesion studies with NMS as the control. Pharmacokinetics profiles and parameters were shown in lability ,39. The The anti-inflammatory pharmacodynamics result was in agreement with in vivo pharmacokinetics result. As can be seen in Tetraethoxysilane  was purchased from Aladdin . Absolute ethyl alcohol was bought from Bodi chemical Co.,Ltd . Methyl alcohol was purchased from Hengxing chemical Co.,Ltd . Aminopropyl triethoxysilane (APTES) was provided by Xiya chemical Co.,Ltd . Octadearyl dimethyl ammonium chloride was bought from Beijing coupling technology Co.,Ltd . Rat pharmacodynamics and anti-inflammatory pharmacodynamics were conducted based on the ethical guidelines that approved by the Ethics Review Committee for Animal Experimentation of Shenyang Pharmaceutical University .CMSN adopted as control carrier was synthesized as follows. 6.6 mg/mL Octadecy trimethyl ammonium bromide (STAB) was prepared using double distilled water and anhydrous ethanol at ambient temperature. In the meantime, 8.547 mmol APTES was added into 15 mg/mL D-tartaric acid ethanol, and the obtained white precipitate (D-APTES) was collected. Subsequently, 68.517 mmol ammonia liquor and 0.8 g D-APTES were added to the template solution under stirring. 17.913 mmol TEOS was dropped into the homogeneous solution under vigorous stirring. After 4 h, the obtained mixture was left statically for 1 day. Lastly, the mixture was washed by water and ethanol and then dried in oven. The template was removed by refluxing in hydrochloric acid methanol solution to get CMSN. E-CMSN was prepared using the above synthesized process except that 488 μL TMB was added into template solution before the adding of ammonia liquor. The schematic diagram of synthetic routes was shown in −1. Samples were produced by grounding with KBr gently and respectively [FTIR  spectra of CMSN and E-CMSN were measured from the spectral region 500 to 4000 cmectively .3mg processed carrier was accurately weighed and mixed with 22 mg KBr uniformly, then the mixture was compressed into tablet using infrared tablet press. Circular dichroism apparatus  was employed to confirm chirality of CMSN and E-CMSN .SEM was obtained with SU8010  to characterize the morphology of CMSN and E-CMSN. Before measurement, samples were mounted onto metal stubs using double-sided adhesive tape and then sputtered with a thin layer of gold under vacuum.The porous structure of CMSNs and E-CMSN was tested using JEM2100 TEM instrument (JEOL. Japan). Sample ethanol solution was withdrawn and then displayed onto porous carbon films.BET), total pore volume (Vt) and pore size distributions, were obtained according to nitrogen adsorption in the relative pressure range from 0.05 to 0.2, the amount adsorbed at a relative pressure of 0.99, and adsorption branches of isotherms respectively.Specific porous structure of CMSN and E-CMSN was studied using V-Sorb 2800P . The major parameters, covering specific surface area  was mixed with 60 mg CMSN or E-CMSN. The system was sealed and stirred gently for 1 day at room temperature. Then the lid was opened with stirring until solvent was evaporated completely to get drug loaded carrier. Finally, unloaded NMS was washed using distilled water . Drug loXRD of a range of samples, covering NMS, CMSN, physical mixture of NMS and CMSN, NMS loaded MSN (NMS/CMSN), E-CMSN, physical mixture of NMS and E-CMSN, as well as NMS loaded E-CMSN (NMS/CMSN), were analyzed using a EMPYREAN XRD . The wavelength was scanned between 5 and 40 °C 2θ with a 0.04° step size.DSC thermograms  of a range of samples, covering NMS, CMSN, physical mixture of NMS and CMSN, NMS/CMSN, E-CMSN, physical mixture of NMS and E-CMSN, as well as NMS/E-CMSN, were obtained at heating temperature from 30 °C to 500 °C at the rate of 10 °C/min.NMS, NMS/CMSN, and NMS/E-CMSN in vitro dissolution were performed in 250 mL dissolution medium (pH 6.8 phosphate buffer solution). The experiment was performed at 37 °C, 50 rpm and time was recorded at the beginning of the experiment. Aliquots (5 mL) were withdrawn at appropriate time intervals and replaced with 5 mL of fresh dissolution medium after each sampling to maintain constant volume . The samTo study the wettability differences after loading NMS into the two carriers, dynamic contact angle measurement was performed using JCY series . The working conditions covered: Automatic contact angle meter model, pressurized tablet of sample, as well as a drop of dissolution medium (2 μL). The measurement was started when dissolution medium dropped onto the tablet ,46.v/v); Column temperature: 30 °C; Analysis wavelength: 370 nm; Flow rate: 1.0 mL/min.9 Male Wistar rats weighing 200 ± 20 g were randomly split into three groups  and fasted for 12 h but given free access to water prior to the experiment. The three samples were suspended in 1.5 mL normal saline with the drug dose of 40 mg/kg and then orally administered (intragastric administration) to rats. Blood media (0.5 mL) of each animal was collected via the suborbital vein at different time intervals after administration and immediately centrifuged at 8000 rpm for 10 min to collect plasma. Subsequently, the obtained plasma was then stored at 20 °C until analysis. 200 μL plasma sample was mixed with 80 μL of an internal standard solution (0.5 mg/mL) and 400 μL acetonitrile. The mixture was vortexed for 2 min. After centrifugation at 10,000 rpm for 5 min, the supernatant of each sample (20 μL) was subjected to HPLC analysis . The wor0 denotes original perimeter of right hind ankle; Ct is the measured perimeter of right hind ankle.Right hind ankle perimeter of rats was ascertained to study the anti-inflammatory biological effect of NMS/CMSNs or NMS/E-CMSN. Twelve male Sprague–Dawley rats (200 ± 20 g) were separated into four groups  and then fasted overnight before experiment. The rats in Group B, Group C and Group D were administered orally with corresponding nanoparticles containing 40 mg/kg NMS, while the rats in Group A were administered orally with normal saline. Afterwards, the perimeters of right hind ankles were ascertained. After half an hour, 0.1 mL 1% carrageenan solution was injected subcutaneously into the paw of each rat. The perimeters of right hind paw ankle after injection were measured at predetermined time intervals . Then, the following parameters were calculated .(2)ReprHealthy Kunming mice were sacrificed after being fastened for 8 h. Then, the small intestine was removed, and the mice were divided into 3–4 segments (4 cm per segment). The inside food residue was rinsed with normal saline. The gel layer was kept on the small intestine and then used in a flat state ,50. Ep t2/g; E-CMSN: 474.360 cm2/g), pore volume  and pore diameter  of E-CMSN were higher than those of CMSN as mesopores were enlarged. After NMS was loaded into CMSN and E-CMSN, most crystalline NMS converted to amorphous phase and E-CMSN was superior, as confirmed by XRD and DSC analysis. The wetting property of NMS and its dissolution were significantly enhanced by the contribution of the two carriers. The anti-inflammatory pharmacodynamics and in vivo pharmacokinetics results were consistent with the wetting property and in vitro drug dissolution results, proving that NMS/E-CMSN exhibited superior NMS delivery system based on its higher oral relative bioavailability and anti-inflammatory effect since its enlarge mesopores contributed to load more amorphous NMS. It is considered that E-CMSN with superior NMS delivery performance will significantly expand its development and bring huge value for the design of novel drug delivery systems.In this study, CMSN and E-CMSN with a particle size from 200 to 300 nm were synthesized. The surface area (CMSN: 440.666 cm


GhTIE1 has not been studied in cotton. Here, we demonstrated that GhTIE1 is functionally conserved in controlling shoot branching in cotton and Arabidopsis. Overexpression of GhTIE1 in Arabidopsis leads to higher bud vigor and more branches, while silencing GhTIE1 in cotton reduced bud activity and increased branching inhibition. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays showed that GhTIE1 directly interacted with subclass II TCPs  in vivo and in vitro. Overexpression of GhBRC1, GhBRC2, and GhTCP13 in mutant brc1-2 partially rescued the mutant phenotype and decreased the number of branches, showing that these TCPs are functionally redundant in controlling branching. A transient dual-luciferase reporter assay indicated that GhTIE1 repressed the protein activity of GhBRC1 and GhTCP13, and thereby decreased the expression of their target gene GhHB21. Gene expression level analysis in GhTIE1-overexpressed and silenced plants also proved that GhTIE1 regulated shoot branching via repressing the activity of BRC1, HB21, HB40, and HB53. Our data reveals that shoot branching can be controlled via modulation of the activity of the TIE1 and TCP proteins and provides a theoretical basis for cultivating cotton varieties with ideal plant types.Transcription factors (TFs) and transcriptional regulators are important switches in transcriptional networks. In recent years, the transcriptional regulator TIE1 (TCP interactor containing EAR motif protein 1) was identified as a nuclear transcriptional repressor which regulates leaf development and controls branch development. However, the function and regulatory network of  Branch development plays a decisive role in controlling aboveground plants, and the aboveground parts of crops are closely related to growth and yield . The devTCP (TEOSINTE BRANCHED1 (TB1) from maize, BRC1 from Arabidopsis, and PROLIFERATING CELL FACTOR from rice) family TFs regulating branch development have been successively discovered. TB1 was first found to be a key gene controlling maize branching  , and the expression level decreased with the growth of buds  , pea PsB  21, HB40, and HB53—which are directly regulated by BRC1 in Arabidopsis , and grown with regular watering under adjusted conditions of 27/20°C, 14/10 h, and humidity of 75%.The cotton materials used in this study were Arabidopsisthaliana ecotype Columbia-0 (Col-0) was used in this study. The mutant brc1-2 was obtained from the laboratory of Dr Genji Qin. The Arabidopsis seeds were sterilized by commercially diluted  NaOCl, followed by several rinses with sterile water. Germination was carried out on sterile Murashige and Skoog (MS) medium. After 7 days, seedlings were transferred to soil and grown at 22°C under long-day conditions .A. thaliana, we used the rosette disc with a bit of the stem and then extracted RNA using the RNAprep Pure Plant Kit (Tiangen). We then used the PrimeScript RT reagent Kit with gDNA Eraser (Takara) to produce cDNAs.We obtained the apical new tissue of cotton, including stem, leaves, and axillary buds. For –∆∆Ct method to calculate the expression level of each target gene  with SYBR Premix Ex Taq  on an ABI 7900 qRT-PCR System (Applied Biosystems). A three-step method was used with the following PCR conditions: 40 cycles at 95°C for 30 s, 95°C for 5 s, and 60°C for 30 s. We checked the dissociation curves of each reaction and used the cycle threshold (CT) 2get gene . Each reAgrobacterium tumefaciens strain GV3101 using the freeze–thaw method.In our study, we constructed all vectors using a one-step cloning method. This method uses a primer with a homologous arm and a double restriction site to amplify the gene fragment, which is then ligated to the vector with the same homology arm and restriction site. All vectors were converted to TIE1-F/R and 35S-BRC1-F/R . Localization of GhBRC1, GhBRC2, and GhTCP13 was obtained using the same method. Sequences of the genes in this study were retrieved from the CottonGen database (https://www.cottongen.org/)  .TIE1 with BRC1, BRC2, and GhTCP13, we amplified the coding sequences for TIE1 and BRC1 and for BRC2 and GhTCP13 using BDTIE1 F/R, ADBRC1-F/R, ADBRC2-F/R, and ADTCP13-F/R, respectively  without Leu and Trp and glucose-free medium (SD/-4) without Leu, Trp, His, and Ade were used for culturing all transformed yeast cells.To examine the interaction of GhBRC1 were fused to the fragment encoding the C-terminus of YFP, and the fragments encoding GhTIE1 were fused to the fragment encoding the N-terminus of YFP, then transformed into Agrobacterium strain GV3101. GV3101 cultures harboring constructs expressing nYFP fusion proteins and cYFP fusion proteins were mixed at a ratio of 1:1 and introduced into N. benthamiana leaves through infiltration. YFP signals were detected by confocal microscopy as described above. For the luciferase (LUC) assay, a 1,500-bp promoter region of GhHB21 was constructed into a vector pGWB435 containing a Luc reporter gene to construct the reporter GhHB21pro-LUC. GhHB21pro-LUC and 35S-pCAMBIA2300-GFP were co-infiltrated into N. benthamiana leaves as a positive control; GhHB21pro-LUC, 35S-pCAMBIA2300-GhBRC1, and 35S-pCAMBIA2300-GhTIE1 were co-infiltrated into N. benthamiana leaves to confirm whether GhTIE1 represses the GhBRC1 activity; and 35S-pCAMBIA2300-GhTCP13, GhHB21pro-LUC, and 35S-pCAMBIA2300-GhTIE1 were co-infiltrated into N. benthamiana leaves to detect the interaction between TIE1 and BRC1 (N. benthamiana leaves were sprayed with luciferin (100 mM) and kept in the dark for 10 min. The leaves were observed under a low-light cooled charge-coupled device (CCD) imaging apparatus Lumazone_1300B (Roper Bioscience) and then photos were taken. Quantitative analysis was done using a Tanon 5200 Multi chemiluminescent imaging system.The vectors pXY104 and pXY106 were used to produce constructs for the bimolecular fluorescence complementation (BiFC) assays, and they carried fragments encoding the C- and N-terminal halves of YFP (cYFP and nYFP), respectively . The cDNand BRC1 . The plaAtTIE1 amino acid sequence as a query sequence, we searched the G. hirsutum protein database using the blastp program (https://www.cottongen.org/) to obtain two homologous genes located in At (GH_A05G0254) and Dt (GH_D05G0259) subgenomes, respectively, named GhTIE1a and GhTIE1d , and the C-terminal region contains a typical EAR motif sequence (DLELRL), showing that GhTIE1a and GhTIE1d may possess a similar function to AtTIE1 as a transcriptional repressor  in 35-day-old seedlings, where there was no observed rosette-leaf branch in WT. Most rosette-leaf buds remained small or dormant. Almost all the rosette-leaf buds in overexpressed (OE) lines grew out to form rosette-leaf branches, and more than four rosette-leaf branches were observed in OE lines , GhTCP13, and GhBRC2 (TCP12), potentially interacted with GhTIE1. The protein blast results and phylogenetic analysis showed that four GhBRC1 proteins were homologous to AtBRC1 and two GhBRC2 proteins were homologous to AtBRC2  and GhBRC2 (GH_A12G1898 and GH_D12G1898) and one copy of GhTCP13 (GH_A05G3929) interacted with GhTIE1d  to construct the reporter construct GhHB21pro-LUC. Then, 35S-GFP as control, 35S-GhBRC1, and 35S-GhTIE1 were co-infiltrated into N. benthamiana leaves with GhHB21pro-LUC. LUC activity analysis indicated that GhBRC1 directly activated the GhHB21pro-LUC. In contrast, when GhHB21pro-LUC and 35S-GhBRC1 were co-infiltrated with 35S-GhTIE1, the activity of GhHB21pro-LUC was inhibited, showing that GhTIE1 represses GhBRC1 transcriptional activity . The possible explanation was that there were four copies of GhBRC1 (AtBRC1. Only two copies including GH_A11G0062 and GH_D11G0067 were silenced in our study. The other two copies of GhBRC1 (GH_A12G2854 and GH_D12G2879) and the two copies of GhBRC2 and GhTCP13 were not simultaneously silenced, which may play a redundant function in regulating branch number.To study the function of HB21, HB40, and HB53 in Arabidopsis are directly regulated by AtBRC1, and AtTIE1 also indirectly repressed the transcription of these genes , GhTCP12 (GhBRC2), and GhTCP13, and these GhTCPs were both grouped into class II. GhTCP18 and GhTCP12 were categorized into TB1 and CYC-like TCPs, respectively. GhTCP13 was categorized into CIN-like TCPs  activity  have strong interactions in vivo and in vitro, and these TCPs both partially rescued the phenotype of brc1 mutant in Arabidopsis. Previous researches found that CIN-type genes limit cell proliferation at the margins of the developing leaf primordia and regulate leave morphology in Antirrhinum, Arabidopsis, and tomato .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The aim of this study was to evaluate the risk of development of colorectal adenomas in patients with colorectal cancer (CRC) with and without colonic diverticulosis.  We performed a retrospective cohort study that included patients with CRC between 2008 and 2011. All patients underwent preoperative colonoscopic and barium enema examinations. Follow-up colonoscopic examinations were performed within 1 year and between 3 and 5 years postoperatively. The incidence of colorectal adenomas was compared based on the presence or absence of diverticulosis. Additionally, multivariate logistic regression analysis was performed to identify the factors independently associated with the development of synchronous and metachronous colorectal adenomas. P > 0.001). Multivariate regression analysis showed that colonic diverticulosis (odds ratio (OR) 3.874, 95% confidence interval (CI) 1.843–8.144, P > 0.001) and obesity  were associated with an increased risk of synchronous colorectal adenomas. The presence of synchronous colorectal adenomas increased the risk of metachronous colorectal adenomas .  Of the 168 patients with CRC included in the study, 55 showed colonic diverticulosis. Synchronous colorectal adenomas were more common in CRC patients with diverticulosis than in those without diverticulosis ( Colonic diverticulosis was associated with synchronous colorectal adenomas in patients with CRC, which is eventually increasing the risk of metachronous adenomas. In Western countries, diverticulosis predominantly affects the left-sided colon and is considered an acquired disease. The prevalence of colonic diverticulosis is <10% in the population aged <40 years and increases with age such that approximately two-thirds of the population aged >60 years is affected , 3. ColoBarium enema examination is performed to detect colorectal abnormalities including diverticulosis, abnormal colonic movements, colonic narrowing or dilatation, polyps, and cancers. Colonoscopic examination is preferred for the detection and removal of colonic polyps and serves as a standard CRC screening tool. Despite the high diagnostic accuracy, colonoscopic examination could miss left-sided colonic diverticulosis, identified by a barium enema study .We performed both colonoscopic and barium enema examinations to detect colonic diverticulosis in consideration of the false negative causes by one diagnostic method. We evaluated the risk of colorectal adenomas in patients with CRC and investigated the association between colonic diverticulosis and adenomas.We performed a retrospective cohort study that included 235 patients who underwent surgical treatment between January 2008 and January 2011 at the Catholic University of Korea, St. Vincent's Hospital. This study was approved by the Institutional Review Board (VC18RESI0175) of the hospital. We enrolled patients who preoperatively underwent both colonoscopic and barium enema examinations for the preoperative detection of synchronous lesions. Patients with CRC were classified into 2 groups—patients with and without diverticulosis. The exclusion criteria were patients aged >85 years and <40 years, patients with advanced-stage cancer (≥stage 4b), patients reporting an incomplete study including failure of endoscopic evaluation for the proximal colon and an incomplete/inconclusive barium enema examination, patients with a history of undergoing screening colonoscopy, patients lost to follow-up, and patients reporting current intake of nonsteroidal anti-inflammatory drugs and low-dose (≤100 mg) and high-dose acetylsalicylic acid.Colonoscopic and barium enema examinations were performed preoperatively. Colonoscopies were performed by 5 board-certified colonoscopists using a standard video colonoscope . All patients were administered polyethylene glycol-electrolyte powder  mixed with 4 liters of water. Colonoscopic examination was performed to evaluate the entire colon for synchronous cancers, adenomas, and diverticulosis. The number, size, and histopathological findings of CRC and colorectal polyps were recorded. Multiple colorectal adenomas were defined as the presence of ≥3 adenomas. Advanced colorectal adenomas were defined as the presence of at least 1 adenoma showing at least 1 of the following features: a colorectal adenoma measuring >1 cm in diameter, tubulovillous or villous adenomas, and/or severely dysplastic lesions. Diverticulosis was defined as the presence of ≥1 diverticula. Based on the locations of diverticula, patients were classified into 3 groups: those with right-sided, left-sided, and bilateral type of diverticular disease. Barium enema examination was performed after the preoperative colonoscopy. The presence, location, and the number of diverticula were recorded by the radiologist. These 2 tests were performed blindly for the examiners, and colonic diverticulosis was detected in one or more positive results. The incidence rates of colonoscopically detected colorectal adenomas were evaluated at specific time points: preoperatively within 1 year and between 3 and 5 years postoperatively.Colonoscopic examinations were performed within 1 year and between 3 and 5 years postoperatively. Adenomas detected by colonoscopic examination within 1 year postoperatively were categorized as synchronous adenomas. Follow-up colonoscopic examinations were performed between 3 and 5 years postoperatively for the evaluation of metachronous colorectal adenomas.t-test. Categorical variables are expressed as quantities and were analyzed using the χ2 test or the Fisher exact test. Logistic regression analysis was used to identify the factors independently associated with the development of colorectal adenomas. All statistical analyses were two-sided, and P < 0.05 was considered statistically significant. We used the SPSS statistical package  for all analyses.Continuous data are expressed as the mean ± standard deviation and were analyzed using an independent sample P = 1.000). Among 55 patients with diverticulosis, 41 (74.5%) patients had right side colon diverticulosis (This study included 168 patients. The mean age of patients was 63.7 years (range 43–3e years), and the mean follow-up was 76.5 months (range 12–ean months). CRC was predominantly located in the left-sided colon: rectum  and sigmoid colon . Of 168 patients, 55 (32.7 %) were classified as the diverticular group and 113 (67.2 %) as the nondiverticular group . No staticulosis . Eighty-Of the 55 patients diagnosed with diverticulosis, 11 patients were diagnosed by both barium enema and colonoscopy. The diagnostic accuracy of colonoscopic examination vs. barium enema examination in detecting colonic diverticulosis was 29.1% (16/55) vs. 90.9% (50/55). Based on the location of the diverticula, patients were classified into 3 groups: those with right-sided, those with left-sided, and those with bilateral type diverticular disease. Right-sided colonic diverticula were observed in 41 (74.5 %), left-sided colonic diverticula in 7 (12.7%), and bilateral colonic diverticula in 7 (12.7%) patients .P > 0.001). Multivariate regression analysis showed that colonic diverticula (odds ratio (OR) 3.874, 95% confidence interval (CI) 1.843–1.843, P > 0.001) and obesity  were associated with an increased risk of synchronous colorectal adenomas.We analyzed 89 synchronous colorectal adenomas which included preoperatively diagnosed colorectal adenomas and those diagnosed by colonoscopic examination within a year postoperatively. Logistic regression analysis was performed to investigate the risk for synchronous colorectal adenomas . SynchroP > 0.001)  and 23.5% (25/106). Univariate analysis and multivariate regression analysis showed that synchronous colorectal adenomas increased the risk of metachronous colorectal adenomas  .This study showed that colonic diverticulosis and obesity are significantly associated with the risk of synchronous colorectal adenomas. Additionally, synchronous colorectal adenomas showed a significant association with the risk of metachronous colorectal adenomas. Previous studies have reported that the incidence of synchronous and metachronous CRCs was 1.8–19.0% and 0.5–3.6%, respectively –10 Most Our results showed that colonic diverticulosis and obesity increased the risk of development of synchronous colorectal adenomas. The association between colonic neoplasms and obesity was similar to previous studies , 15. HowColonic neoplasia and diverticulosis share common epidemiological and pathological characteristics. A potential hypothesis that explains the association between colonic neoplasia and diverticulosis is that both diseases are linked to chronic inflammation of the colonic mucosa , 25. PreMultivariate analysis did not show any association between metachronous colorectal adenomas and colonic diverticulosis. Nevertheless, because metachronous adenomas are eventually associated with synchronous polyp, it is likely that metachronous adenoma will increase if there is colonic diverticulosis. A recent meta-analysis assessing the risk factors for metachronous CRC or polyps showed that the presence of synchronous colonic polyps served as a risk factor for metachronous lesions, which concurs with our results . TherefoColonoscopic examination scores over other screening tests in the accurate detection of colonic polyps and CRC. However, it shows limitations in the evaluation of colonic diverticula . EndoscoThis study is the first to use 2 screening modalities to establish an accurate diagnosis of colonic neoplasia and diverticulosis. We observed that approximately one-third of the patients diagnosed with CRC showed colonic diverticula. Among these, only 29% of patients were diagnosed based on colonoscopic examination indicating the high probability of missing a significant number of diverticula if only colonoscopic examination was to be performed for the diagnosis of diverticula.This study has several potential limitations. First, we cannot ignore the possibility that a selection bias may have influenced the results owing to the retrospective design. Second, approximately one-third of the included patients were lost to follow-up and did not undergo colonoscopic examination 3–5 years postoperatively. Finally, these results cannot be applied to patients in Western countries because this is a single-institution study performed in Asia. Nevertheless, this is a useful study that accurately diagnosed diverticulosis and analyzed its relationship with adenomas.In conclusion, CRC patients with colonic diverticulosis showed a significantly high risk of synchronous colorectal adenomas. Additionally, synchronous colorectal adenomas increased the risk of metachronous colorectal adenomas. Therefore, meticulous colonic examination is warranted in patients with CRC and concomitant diverticulosis.