Objective: The ear irritations suffered by hearing aid (HA) users are yet to be related to the clinical state of canal. We undertook this study to examine the nature of debris and the microbial flora of ears of hearing aid users, as well as evaluate the determinant factors of ear irritation in this population.Methods: An observational clinical study was carried out involving 32 unilateral hearing aid users recruited from ENT clinic of a tertiary referral center. Each subject underwent otoscopic assessment of canal debris and microbial analysis of swab cultures taken from the hearing aid-wearing ear and contralateral normal ear without hearing aid.Results: Canal debris . as well as microorganisms were identified in significant number of ears with hearing aids than ears without hearing aid (P = 0.003 and P = 0.006 respectively). Coagulase-negative staphylococci were the commonest identified bacteria. Others were Staphylococcus aureus, Pseudomonas aeruginosa, and Proteus species. Intolerable irritations of hearing aid wearing ears were significantly associated with bacterial and fungal otitis externa, and ear discharge .Conclusions: This study demonstrates that using hearing aid alters the ear canal flora; increases risk of both fungal and bacterial otitis externa, as well as encourage wax debris formation, with resultant ear irritations. To ensure compliance their ears should periodically be attended to, by de-waxing or given topical antimicrobial agents where indicated. The irritation, itching, and discharge from the ear canal usually present their challenges that sometimes make the use of HA difficult or even impossible. Some HA users get easily dissatisfied with the use of the aids regardless of the hearing benefits they derive. Some other users simply discontinue the use of the hearing aids on account of the irritations from debris accumulation in the canal. The most challenging scenario is with persistent bacteria/fungal otitis externa where the otolaryngologists sometimes consider fitting of the aids via more invasive alternative routes such as middle ear hearing implants.5-7 A wide range of contaminating bacteria and fungi growing on hearing aid surfaces as well as ear canals of HA users has been documented.3,4,8,9 However the growths of these microorganisms are yet to be related to the clinical state of canal with HA with and whether these organisms result in actual infection of the external auditory canals.Hearing aid (HA) users often present to otolaryngologist with complaints of constant irritations within the ear canals on account of either allergic contact dermatitis from the earmolds which connect the HA to the ear canal,This study was carried out to examine the characteristics and microbial profile of ear canal debris, and to study the determinant factors of ear irritation in hearing aid users.Thirty two hearing impaired individuals previously fitted with behind the ear HA, who arrived at the Ear nose and throat clinic of a tertiary health institution for routine hearing aid follow-up, or for HA related or some other complaints participated in this study.Potential subjects were informed of the proposed study and asked to volunteer. Once subjects had given informed consent, their eligibility for inclusion was further evaluated. The approval of the study was given by the Institutional Ethical Review Committee.These volunteers included individuals who were fitted with HAs in our department as well as those who were fitted elsewhere before presenting to our department. All the subjects were using the behind-the-ear HA. One was recruited if he/she has used a uni-aural HA for upwards of four weeks. Volunteers were excluded if they had active ear discharge originating from the middle ear in the presence of tympanic membrane perforation, or had history of penetrating trauma to the ipsilateral ear, or had taken any antimicrobial ear drops or systemic antibiotics in the preceding seven days.On arrival at the clinic, potential subjects were interviewed and data concerning the age, duration of HA usage, history and frequency of ear irritation, pain, and discharge were enquired. The irritation was regarded as mild if occurs occasionally; moderate if it occurs almost all the time; severe if it is associated with ear pain, or discharge in the absence of any tympanic membrane perforation, or resulted in discontinuation of the use of HA. The ears were examined with otoscopes and the states of the ear canals and the ear drums recorded. Any debris/exudates seen were characterised. The ear canals were classified as either normal appearance; or as having otomycotic debris if it reveals fungal deposits/exudates; or bacterial exudates if there is purulent exudates; or wax debris; or a combination of the various debris.10,11Each volunteer had two ear swab samples taken, one each from the hearing aid-wearing ear canal and the other from the contralateral ear canal regardless of whether debris/exudates were found or not provided the tympanic membrane was intact. The samples from the contralateral ear served as the control. The samples were taken before cleaning the ear canals of any debris/exudates or cerumen, with sterile swab sticks which were enclosed in air tight plastic tubing and then transported to the microbiology test laboratory. The swabs were plated on MacConkey agar plates and incubated for 48 hours after which bacterial isolates and the antimicrobial sensitivities were identified using standard microbiological methods.Statistical Analysis: The Statistical Package for Social Sciences  version 16 was used for analysis. Chi square test was used to test the statistical significance of association between potential variables.A total of 64 ear swab samples, obtained from 32 hearing aid users, were analyzed. Seventeen male and 15 female patients  were included in the study. There were complaints of irritations in 18 (56%) of the HA wearing ears and in 3 (9%) ears without HA. There were also complaints of pain  and discharge  in those ears in which a HA was worn.2 = 12.67; p = 0.003). Although ear wax accumulation seem slightly more identifiable in HA wearing ears compared to the contralateral ears without HA, the difference was not significant .2 = 8.60; p = 0.006). Regarding the details of the microbial isolates from the HA wearing ears, mixed bacterial and fungal isolates of Proteus and Candida spps. were identified in one of the ear swabs. Coagulase-negative staphylococci were isolated in five of the HA wearing ears and two ears without HA where otoscopy earlier revealed either no debris or wax. The rest of the cultures yielded other bacteria shown in The microbial cultures of the swabs from the HA wearing ears identified 9 bacteria and 7 fungal isolates compared to the significantly fewer isolates from ears without HA . In 2 = 30.49; p = 0.002).  analysis of the association of different potential factors in relation to non-tolerance of the HA occasioned by severe ear irritations among the 32 HA users. Strong significant connections were found between severe intolerable ear irritations and presence of ear discharge, fungal ear infection, as well as bacteria otitis externa .Ear irritations were significantly documented more in the ears with HA (21), compared to three ears without HA  than ears without HA (41%).8,13 In this present study they were cultured in ears that showed no clinical evidence of otitis externa, but more in ears with HA than those without HA. This seems to suggest that HA encourages the proliferation of not only pathogenic bacteria causing otitis externa, but also non-pathogenic bacteria with potential for producing ear irritation.Coagulase-negative staphylococci are usually considered to be non-pathogenic and their role in otitis externa is still largely unknown. They have been cultured in the normal external auditory canal, either alone or in combination with other organisms, including diphtheroids or occasional fungal sporesP = 0.003). It is therefore conceivable that hearing aid mould increases the tendency for ear canal irritations by encouraging proliferation of both pathogenic and non-pathogenic microorganisms, as well as accumulation of wax debris. However, the ear irritations were more severe in the presence of bacteria and/or fungal infections than in ears with only wax debris.Ear irritations were documented in significant numbers of the ears with HA compared to contralateral ears without HA in this study. Furthermore, in the ears with HA, 89% of these irritations were noted in ears where debris were identified in the canal, including wax as was outlined in Although fungal otitis externa was significantly associated with intolerable irritations in this study, the data indicated that bacteria otitis externa constitute the most significant factor for discontinuation of HA usage due to resulting severe intolerable ear irritation. Bacterial infections are obviously likely to be associated with inflammation of the ear canal, and are understandably more likely to result in ear pain as well as discharge, which will make the continued use of HA difficult or perhaps impossible. Users that experience both ear pain and discharge resulting from bacteria otitis externa will most likely discontinue the HA pending their resolutions.3,8,9 It has been demonstrated that Cleaning with 70 per cent alcohol solution was ineffective, in particular for pathogenic fungi on ear moulds.4Some of the subjects in this study justifiably blamed their HA for the irritations and ordeal they underwent, and often contemplated the option of abandonment of the HA. It is therefore instructive for periodic attention to be paid to the care of their ears, by de-waxing or given topical antimicrobial agents where indicated, as part of measures to improve the comfort and ensure compliance of HA usage. Some authors have recommended education of HA users in the use of an appropriate hygiene routine to clean and disinfect hearing aids and ear moulds in order to avoid otitis externa, by wiping the surfaces with a tissue soaked in a non-alcohol-based disinfectant which should be stored in an appropriate case for later use.1,2 In one particular series, contact allergy to the ear mould material was found in 27% of the patients, where a positive test reaction were documented to methyl methacrylate, triethyleneglycol dimethacrylate and urethane di-methacrylate skin patches.1It is of interest to observe that not all the ear irritations documented in this study resulted from wax or infective debris as two of the patients reported irritations in their HA wearing ears in absence of any documented debris in the canal on otoscopy. The irritation was even so severe to the extent that the use of HA was discontinued in one of them. The patient was only able to recommence the use of the HA after the resolution of the troublesome irritation following use of oral doses of antihistamine and steroid-based ear drop. It seems reasonable to believe that the irritations in these two cases were occasioned by contact allergic reaction to the HA ear moulds, or due to mechanical pressure exerted on the lining of the canal by the ear mould. Ear irritations due to contact allergic reactions to HA ear moulds have been reported.In conclusion, wearing HA seems to modify the microbial flora of the ear canal. It encourages proliferation of not only the commonest non pathogenic coagulase negative Staphylococci, but also the pathogenic bacteria and thereby increases the risk of otitis externa. Although the resulting ear irritations seem to be most severe in the presence of bacteria as well as fungal otitis externa, it is also significantly observed with wax debris in the canal. Finally, the possibility of contact allergic dermatitis developing due to sensitization of the ear canal to ear mould material should be considered in HA users presenting with severe ear irritation in the absence of obvious ear canal debris.


Telocytes, a novel type of interstitial cells with very long and thin prolongations, have been identified in many organs in mammals. At present, the ultrastructural, immunocytochemical and electrophysiological properties of telocytes in multiple organs have been understood. However, telocytes in spleen, especially their roles in spleen have not been reported. The aim of this study was to investigate the ultrastructure, distribution and immunophenotypes of splenic telocytes. Rat spleen was harvested for the ultrastructure analysis by transmission electron microscopy (TEM). The primary culture of telocytes was performed after combined enzymatic digestion. The characteristic morphology was analyzed by a scanning electron microscopy (SEM). It was shown that telocytes displayed a piriform/spindle/triangular shape with long and slender telopods and extremely long prolongation contracting with surrounding cells in the spleen. Their dynamic profiles of cytoplasmic separation were recorded by the Live Cell Imaging System. The length of telopods was mostly distributing in 20–30 μm, in accordance with normal distribution. Most telocytes had three or two telopods (28.71% and 22.58% respectively). Immunostaining indicated that these cells were positive for vimentin, CD34, nanog and sca-1, but negative for c-kit. These data prove the existence of telocytes in the spleen, which may serve as the experimental base for exploring their roles in the spleen. As a new type of interstitial cells, telocytes(TCs) were detected in various tissues/organs in mammals, such as heart ,2, skeleet al. reported that TCs represented about 1–1.5% of the atrial myocardial volume and presumed their potential role in interstitium, such as players in pacemaking, intermediates between intrinsic nerve fibers and myocytes, a paracrine and/or juxtacrine source of signal molecules  and approved by the Xinxiang Medical University Animal Care Committee (No 030032).Four male Sprague-Dawley rats and twenty neonatal Sprague-Dawley rats  were utilized in this study. Animal treatment was performed according to the guidelines of 3 and immediately immersed in a solution of 4% glutaraldehyde . Fixed samples were washed in phosphate buffer, and were fixed in 1% osmium tetroxide  for 1 hr. Samples were then rinsed extensively in 0.1 M cacodylate buffer. Following several rinses, samples were dehydrated in a graded series of ethanol and were embedded in Epon 812 resin . The embedded samples were heat dried at different temperatures . Then sections of 50 nm were cut with a Leica Ultracut UCT ultramicrotome , stained with 3% solution of uranyl acetate and lead citrate, and mounted on formvar-coated 50 mesh grids. Digital pictures were obtained and observed at an acceleration voltage of 80 kV, in JEOL JEM-1230 (Japan) electron microscope.Male adult rats were euthanized with chloral hydrate  by peritoneal injection. The spleens were harvested and cut into small pieces about 1 mm3 after washing with sterile phosphate-buffered saline (PBS), then incubated at 37°C for 6 min with 0.1% collagenase I/0.125% trypsin  in PBS. The suspension was collected and added into an equal volume of dulbecco minimum essential medium–low glucose (DMEM-LG), supplemented with 10% fetal calf serum  to inactivate the role of combined enzymes. This process was repeated for 2–3 times until the tissue blocks were completely digested. And then, the suspension was centrifuged at 1,000 r/min for 5 min. The cellular pellet was re-suspended in DMEM-LG culture medium supplemented with 10% fetal calf serum, 100 U/ml penicillin and 100 mg/ml streptomycin (Sigma-Aldrich). Cell density was counted in a haemocytometer and viability was assessed by Trypan blue staining. The cells were placed in 24-well plates and cultured in a CO2 incubator at 37°C. After 45 min, suspensions were plated in 24-well plates to remove fibroblasts. Culture medium was changed every 48 hrs. The morphology of TCs were observed and imaged with a phase-contrast microscope .Samples from neonatal rat spleens were harvested under sterile conditions and mechanically minced into small pieces of blocks about 1 mmTCs were fixed in 3% glutaraldehyde in PBS (pH 7.2) for 30 min at room temperature, then dehydrated through graded ethanol  and preprocessed with vacuum deposition of gold plating, which were imaged by a FEI Quanta200 environmental scanning electron microscope .TCs were plated in dish and cultured in CO2 incubator. When cells reached 50%-60% confluence, the medium was changed with DMEM-LG medium containing 0.05 mg/L 4', 6—diamidino-2-phenylindole (DAPI) in a CO2 incubator at 37°C about 40 min, and then the supernatant was discarded to remove the unbinding of DAPI, subsequently washed with PBS. TCs cultured in complete DMEM-LG medium were detected under a Live Cell Imaging System . Parameters were set as follows: 1 image/2 min, duration time 24 hrs.The cultured cells were fixed in 4% paraformaldehyde for 20 min at room temperature, subsequently washed with PBS and treated with 0.3% Triton X-100(Sigma) in PBS for 15 min. Then the samples were incubated with PBS containing 2% bovine serum albumin (BSA) at 37°C for 10 min. Incubation with the primary antibodies was performed at 4°C overnight. The samples were subsequently incubated FITC- or Cy3-conjugated secondary antibodies (Beyotime). The nuclei were counterstained with 1μg /ml DAPI (Roche). Negative controls were obtained by following all the same protocol but the primary antibodies. All experiments were performed in triplicate.P < 0.05 was considered statistically significant.Data were presented as mean ± SD. One-way (ANOVA) and the LSD tests were applied using SPSS version 19.0. In spleen, TCs with long telopode (TP) were seen clearly in red pulp. They connected with leukocytes and red blood cells through TP. TPs were detected mostly basing on their discontinuous segments with alternation of podom and podomer by TEM. TCs embraced with one or more leukocytes, forming a network structure or labyrinthine structure . The nucin vitro. After plating the TCs adhere to the flasks in 60–90 min, attached TCs exhibited a small prominent body and their thin and long TPs after 44–48 hrs of incubation . These Tps have an alternation of very thin segments named podomers and dilated portions named podoms ,3,4. Almet al. described these cells and had many comprehensive researches on TCs. They insisted on that no specific markers had been yet defined for TCs [in situ and in vitro [Popescu  for TCs ,4. In th for TCs , 24. Car for TCs . Cardiac for TCs . In skelin vitro , 27. Butin vitro . Howeverin vitro , 29. Nanin vitro , 31. MarSo far, the exact function of TCs has not been fully understood. Cardiac TCs were detected in stem cell niche in the vicinity of stem and progenitor cells , 17, 32.Taken together, we have investigated the existence, characteristics, and distribution of TCs in the spleen and have detected the immunophenotypes of cultured TCs from the spleen, which could provide direct morphology evidence for exploring the exact role of TCs in spleen.


P < 0.05). The potential mechanism of AK027294 might be associated with the regulation of caspase-3, caspase-8, Bcl-2, MMP12, MMP9, and TWIST. The lncRNA expression profile in colorectal cancer suggests lncRNAs may play important roles in the occurrence and progression of colorectal cancer. AK027294 is highly expressed in colorectal cancer and closely correlates with colorectal cells proliferation, migration, and apoptosis.This study is aimed to investigate the differentially expressed long non-coding RNAs (lncRNAs) in colorectal cancer and its potential biological function. Colorectal adenoma is the precancerous lesions of colorectal cancer, so in this study, we used colorectal adenoma as negative control. The global lncRNA expression profile in colorectal cancer and adenoma was evaluated by bioinformatics. The biological functions and potential mechanism of AK027294 were investigated in HCT116, HCT8, and SW480 colorectal cancer cells. A total of 135 lncRNAs were found to be differentially expressed in colorectal cancer and adenoma tissues. Among them, 71 lncRNAs were up-regulated and 64 lncRNAs were down-regulated. Especially, AK027294 was found to be highly expressed in colorectal cancer tissues compared with colorectal adenoma tissues (fold change is 184.5). Our results indicated that AK027294 down-regulation significantly inhibited colorectal cancer cells proliferation and migration, but promoted cell apoptosis (The online version of this article (doi:10.1007/s13277-015-4350-x) contains supplementary material, which is available to authorized users. Colorectal cancer (CRC) is the third most common cancer in the world, with an estimated incidence of more than one million case , 2. AlthLong non-coding RNAs (lncRNAs), sized from 200 nt to 100 kb, is reported to be vital for governing fundamental biological processes, such as cell proliferation, cell migration, cell cycle, and cell apoptosis –6. HowevIn this study, firstly we explored the differentially expressed lncRNAs in colorectal cancer tissue compared with colorectal adenoma tissues by using bioinformatics, then we screened out the most differentially expressed lncRNAs with biggest fold change. After this, we further detected the biological functions and potential mechanism of the screen out lncRNAs, for instance, the effects of the screen out lncRNAs on the proliferation, migration and apoptosis of colorectal adenoma cells. It may provide a promising target gene to prevent the development and progression of colorectal cancer.http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE37364). GSE37360 microarray information: total RNA was extracted from colonic biopsy samples of histologically negative patients and of patients with adenoma or colorectal cancer and were hybridized on Affymetrix HGU133 Plus 2.0 microarrays. Then 5635 lncRNAs gene expression data were obtained  and determined its concentration was determined. Total RNA (0.5 μg) was used as a template to prepare cDNA . The messenger RNA (mRNA) expression of target genes was quantified using SYBR Premix EX Taq  on the ABI 7500 squence detection system . PCR was performed with the following thermocycling conditions: an initial of 5 min at 95 °C, followed by 40 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s. The thermocycler used in the present study was the StepOnePlus™ Real-Time PCR system . All experiments were performed in triplicate. Finally, the 2‑ΔΔCt method was performed to calculate the relative expression. The primers were obtained from Funengbio Co. . The primer sequences are as follows.2 atmosphere at 37 °C.Human CRC cell lines HCT116, HCT8, and SW480 was acquired from the American Type Culture Collection (ATCC) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5 % fetal bovine serum (FBS) in a humidified incubator with 5 % COHCT116 cells were transiently transfected with negative control small interfering RNA siRNA  and AK027294 siRNA (200 nM) , respectively, using lipofectamine 2000 reagent  according to the manufacturer’s instructions. These siRNAs were commercially synthesized by Funengbio Co. . After incubated for 24 h, cells were collected for follow experiments.2 atmosphere at 37 °C. Then the open wound area was measured at 0 and 24 h. The experiments were repeated for three times.HCT116 cells were cultured in 6-well plates and transfected with different oligonucleotides. When growing to about confluent of 90 %, cells were scratched by a sterile 10 μl pipette tip and incubated in a humidified incubator with 5 % CO5) in 100 μl of serum-free DMEM were cultured in 8-μm culture inserts. Then, the lower chamber of the culture inserts was filled with 20 % FBS-DMEM. Cells were cultured for 24 h, fixed with methanol for 30 min, and stained with hematoxylin for 20 min. Finally, migrated cells were counted in five random fields under an inverted microscope (×20).After transfection at 24 h, cells  in accordance with the manufacturer’s instructions. The cell proliferation curves were plotted by the absorbance values at different time point. All experiments were performed for three times.About 2 × 10Total protein was collected using a lysis buffer and quantified by bicinchoninic acid (BCA) method. Cell lysates were separated using 10 % SDS-PAGE and transferred to the polyvinylidene difluoride membrane. The membranes were immersed into PBST solution supplemented with 5 % non-fat milk for 2 h. Then, the membranes were incubated with antibody-caspase 3 , antibody-caspase 8 , antibody-Bcl2 , antibody-TWIST1 , and antibody-GAPDH,, respectively, at 4 °C overnight. All membranes were incubated with appropriate second antibodyat room temperature for 1 h and visualized by enhanced chemiluminescence .5) were collected by using Trypsin-EDTA and washed with PBS. Then, cells were centrifugated and incubated with propidium iodide  and annexin V-FITC  for 15 min at room temperature. Then, cell apoptosis was assessed by flow cytometry. Each test was repeated three times.Cells  arise from colorectal adenomas [135 lncRNAs might be potential biomarkers associated with the formation of CRC. According to the data, we found that 71 and 64 lncRNAs were up-regulated and down-regulated in colorectal cancer, respectively. Then, we focused on AK027294 lncRNA, which was found to be correlated with DNA replication and cycle process in colorectal cancer by the enrichment of signaling pathways.Recent studies have demonstrated that lncRNAs are linked to the formation and progression of CRC –16. Howeadenomas , 18. ThuAK027294 lncRNA, a novel molecular, was found to be evidently higher in colorectal cancer than that in colorectal adenoma. Although enrichment of signaling pathways demonstrated that AK027294 was correlated with DNA replication and cycle process, its potential functions remained elusive. Thus, we investigated the biological function of AK027294 in colorectal cancer cells. Results showed that the down-regulation of AK027294 was able to inhibit proliferation and migration of colorectal cells, and promote apoptosis in colorectal cancer. Howerver, the mechanism of AK027294 was little to be known. It is well known the activation of caspase 3 and caspase 8 is responsible for cell apoptosis , 20. BclThus, we detected the expression of MMP9, MMP12, and TWIST1 in colorectal cancer cells. The results showed that MMP9, MMP12, and TWIST1 were down-regulated when AK027294 expression in colorectal cells was knocked down. These results indicated that the effect of AK027294 down-regulation on cell migration was correlated with MMP9, MMP12, and TWIST1.In summary, we screen a lncRNA expression profile associated with colorectal cancer and colorectal adenoma. AK027294 is confirmed to be a novel lncRNA, which is correlated with cell proliferation, migration, and apoptosis in colorectal cancer cells. Our findings suggest that AK027294 may play an important role in colorectal occurrence and progression and may be a potential target to prevent progression of colorectal cancer. As this study is the first to report the biological functions of AK027294, further detailed and in-depth investigations are needed to validate our observations.ESM 1(DOCX 130 kb)ESM 2(DOCX 160 kb)ESM 3(DOCX 139 kb)ESM 4(XLS 884 kb)ESM 5(XLSX 21 kb)Below is the link to the electronic supplementary material.


Granulin-epithelin precursor (GEP), a secretory growth factor, demonstrated overexpression in various human cancers, however, mechanism remain elusive. Primary liver cancer, hepatocellular carcinoma (HCC), ranks the second in cancer-related death globally. GEP controlled growth, invasion, metastasis and chemo-resistance in liver cancer. Noted that GEP gene locates at 17q21 and the region has been frequently reported to be amplified in subset of HCC. The study aims to investigate if copy number gain would associate with GEP overexpression.Quantitative Microsatellite Analysis (QuMA) was used to quantify the GEP DNA copy number, and fluorescent in situ hybridization (FISH) was performed to consolidate the amplification status. GEP gene copy number, mRNA expression level and clinico-pathological features were analyzed.r = 0.331, P = 0.010). Gain of GEP copy number was observed in 20% (12/60) HCC and associated with hepatitis B virus infection status (P = 0.015). In HCC with increased GEP copy number, tight association between GEP DNA and mRNA levels were observed .GEP DNA copy number determined by QuMA corroborated well with the FISH data, and the gene copy number correlated with the expression levels  contains supplementary material, which is available to authorized users. Granulin-epithelin precursor (GEP) contributes in multiple vital biological processes and its alias partly indicates its function, including progranulin, proepithelin, acrogranin, and PC cell-derived growth factor. GEP is a pluripotent growth factor important in fetal development, neuronal cell survival, wound healing and tumorigenesis -4. Over-The incidence of primary liver cancer, hepatocellular carcinoma (HCC), has been increasing globally in the past two decades. HCC is the second most frequent cause of cancer-related death worldwide . Major rSixty patients underwent curative partial hepatectomy for hepatocellular carcinoma (HCC) between September 2002 and July 2005 at Queen Mary Hospital, Hong Kong, were recruited with informed consent. Clinico-pathological data were prospectively collected. Ten blood samples from healthy individuals were recruited and served as control with informed consent. The study protocol was approved by the Institutional Review Board of the University of Hong Kong / Hospital Authority Hong Kong West Cluster (HKU/HA HKW IRB).Copy number of GEP gene was measured by Quantitative Microsatellite Analysis (QuMA) as described using quantitative real-time PCR amplification . Chromos-ddCT. Tolerance interval (TI) was calculated to determine if the test sample in the QuMA measurement was significantly different from the mean of measurements made on samples from the healthy individuals [where ddCT = dCT (test DNA) – average dCT , and E = PCR efficiency. The primers had showed PCR efficiencies >95% . Continuous variables were assessed by Pearson correlation analyses. The comparison of categorical variables was examined by Pearson chi-square test with Yates continuity correction. Difference was considered statistical significant if the The GEP DNA copy number was measured by QuMA using real-time PCR. The efficiencies of the PCR were examined   by FISH analysis. Both BAC clones (RP11-436 J4 and RP11-52 N13) flanking GEP gene demonstrated increased DNA copy number  .GEP transcript levels had been demonstrated to be significantly elevated in HCC compared with their adjacent non-tumor liver tissues and normal livers from healthy individuals ,15. The P = 0.015)  Table .Table 2Increased GEP transcript and protein levels have been reported in various human cancers -7. The eElevated expressions of growth factors and oncogenes have been reported to associate with gene copy number gain. In the current study, the GEP gene copy number was investigated by real-time PCR based method QuMA and FISH analysis. QuMA has been commonly employed for quantitative measurement of DNA copy number ,36,37. NCopy number gain at specific chromosomal region contributes on activation of oncogenes and growth factors. The process is important during tumor initiation and also progression along carcinogenesis. However, there always have concerns on whether high levels of amplification are necessary or if the gain of single extra copy would be able to advance cancer. Recently, there are reports that low copy number gain contributes on cancer progression -45. GainRecurrent genomic and expression alterations have been reported on chromosomal arm 17q. Independent studies demonstrated expression gains of gene set at 17q12-21  and 17q2These observations show copy number gain of GEP gene at 17q21 in 20% HCC, and the increased GEP gene copy number correlated with enhanced expression levels in these HCC. This partly provides a mechanistic explanation for the over-expression of GEP for the subset of HCC. Future studies should also examine the chromosomal region at 17q21 for the minimal essential set of genes for HCC initiation and progression. Notably, GEP over-expression has been observed in over 70% HCC ,15. Furt


Auditory neuropathy (AN) is a recently recognized hearing disorder characterized by intact outer hair cell function, disrupted auditory nerve synchronization and poor speech perception and recognition. Cochlear implants (CIs) are currently the most promising intervention for improving hearing and speech in individuals with AN. Although previous studies have shown optimistic results, there was large variability concerning benefits of CIs among individuals with AN. The data indicate that different criteria are needed to evaluate the benefit of CIs in these children compared to those with sensorineural hearing loss. We hypothesized that a hierarchic assessment would be more appropriate to evaluate the benefits of cochlear implantation in AN individuals.Eight prelingual children with AN who received unilateral CIs were included in this study. Hearing sensitivity and speech recognition were evaluated pre- and postoperatively within each subject. The efficacy of cochlear implantation was assessed using a stepwise hierarchic evaluation for achieving: (1) effective audibility, (2) improved speech recognition, (3) effective speech, and (4) effective communication.The postoperative hearing and speech performance varied among the subjects. According to the hierarchic assessment, all eight subjects approached the primary level of effective audibility, with an average implanted hearing threshold of 43.8 ± 10.2 dB HL. Five subjects (62.5%) attained the level of improved speech recognition, one (12.5%) reached the level of effective speech, and none of the subjects (0.0%) achieved effective communication.CIs benefit prelingual children with AN to varying extents. A hierarchic evaluation provides a more suitable method to determine the benefits that AN individuals will likely receive from cochlear implantation. Auditory neuropathy (AN) is a recently recognized hearing disorder primarily characterized by disrupted auditory nerve synchronization, intact outer hair cell function, and poor speech recognition –5. AN maet al. [et al. [Cochlear implantation used to be contraindicated for AN due to its association with nerve degeneration secondary to processes such as demyelination and axon impairment . Howeveret al.  and Trau [et al.  reported [et al. –26. Base [et al. , 27. How [et al. , 28–31,  [et al. , 25, 30.Various criteria have been used to evaluate the benefit of CIs in individuals with AN. Researchers have drawn both positive , 17–19 aSpeech recognition is the main functional impairment of AN, and a substantial challenge in clinical management of AN individual is hearing restoration. The most well-established and useful evaluation methods for identifying CI candidates assess the individual’s speech recognition capability. The results of evaluations from before and after implantation can be compared to determine the quantitative changes in speech recognition , 25, 36,Given the diversity in available evaluation methods, a proper criterion for determining the benefits of CIs in AN individuals is needed. This could be accomplished by using a stepwise hierarchic assessment that demonstrates the effects of cochlear implantation by rating categories or capability levels according to various psychophysic and physiologic results. This hierarchic assessment could be based on functional performance, including hearing sensitivity, speech recognition, language skills, communication skills, and even academic or social skills . VariousDiagnostic test sets for AN, including pure tone audiometry, auditory brain stem response (ABR), cochlear microphonic (CM), distortion-product otoacoustic emissions (DPOAEs), tympanometry, and stapedial acoustic reflex, were performed before implantation to determine whether a subject was eligible for inclusion in the study. Visual reinforcement audiometry or play audiometry was used for children too young for the standard pure tone audiometry examination.The following inclusion criteria were used in the present study: (1) prelingual children who underwent cochlear implantation at < 5 years of age; (2) normal tympanic membranes upon otoscopic examination; (3) presence of nonconductive hearing loss according to audiogram and tympanogram results; (4) absent or seriously abnormal ABR waveforms or ABR thresholds that are disproportionate to the pure tone behavior thresholds; (5) presence of DPOAEs or CM, and (6) presence of CM and type A tympanogram in subjects without DPOAEs.The following exclusion criteria were used in the present study: (1) not meeting the aforementioned inclusion criteria; (2) having a CI that had been switched on for no more than 1 mo or a CI that had never been switched on before the data analysis; and (3) loss to follow-up.In total, ten prelingual AN children received CIs in our study. However, two of these children (ANCI_06 and 08) were removed from the study according to the exclusion criteria, and their data were not included in the analysis. Thus, data from eight prelingual children  with AN who met the inclusion criteria were analyzed . All subBecause of the variability among the subjects in their cooperation with completing the hearing and speech test, hearing sensitivity and speech recognition performances of all eight children were individually evaluated preoperatively and postoperatively. Aided hearing thresholds were tested via behavioral audiometry in sound fields using warble tones. ECAP was tested intraoperatively and postoperatively to evaluate neural activity. An ECAP response including both N1 and P1 peaks was considered as a valid ECAP waveform. To quantify the presentation of ECAP, the electrode array was divided into three parts according to the corresponding location in the cochlea after implantation: basal (electrodes #1–7), middle (electrodes #8–15), and apical (electrodes #16+). The ECAP of the subject was considered normal only when a valid ECAP waveform was recorded from at least one electrode in each part.The assessment of speech recognition varied depending on the subject’s age at the time of implantation, hearing–speech experience, and ability to cooperate with the tests, including the Mandarin Early Speech Perception Test (MESP) , 40, MAICommunication ability was reflected by speech production, which was evaluated by the Speech Intelligibility Rating (SIR). SIR scales the speech intelligibility of children into five categories by the parents under the instruction of an audiologist. The lowest level of SIR describes unintelligible connected speech, involving the use of pre-recognizable words, where the primary mode of communication may be manual. The highest level of SIR describes connected speech that is intelligible to all listeners, where the child is easily understood in everyday context , 43.The efficacy of cochlear implantation was evaluated using a stepwise hierarchic assessment for achieving: (1) effective audibility, such that thresholds were significantly improved and/or electrically evoked responses (including ECAP and EABR) were present; (2) improved speech recognition, which means better postoperative speech recognition scores than preoperative ones assessed via either questionnaires (including MAIS/IT-MAIS) or speech audiometry ; (3) effective speech, with improved sentence recognition and/or speech recognition ability in noisy conditions  and EABR (Subject ANCI_02). Five children  who were able to cooperate for behavioral audiometry showed improvement in the thresholds of speech frequency by 30 to 70 dB, with an average postoperative threshold of 43.8 ± 10.2 dB HL. Both children who had used hearing aids demonstrated a 15- to 25-dB advantage with the CI over the hearing aids, particularly at frequencies > 2 kHz .P < 0.01). Four children underwent a MAIS/IT-MAIS after more than one year. Overall postoperative total, detection, and perception scores of MAIS/IT-MAIS for all eight children on the final test session were 66.5 ± 18.4%, 75.1 ± 22.2%, and 61.9 ± 14.7%, respectively, which were significantly higher than the preoperative scores . Seven of the eight children cooperated during performance of the MESP. Two children (ANCI_04 and 05) passed all categories in the standard version. Three children  made progress in the low-verbal version, but could not complete the standard version. The remaining two children (ANCI_01 and 02) did not progress past the first category (“cannot test”) in the low-verbal version , which can be demonstrated through electrophysiologic tests or pure tone audiometry. Speech efficacy is the second level, and is difficult to assess. Two sublevels of speech performance were therefore established, a lower level representing an improvement over the preoperative performance regardless of the absolute speech recognition ability, and a higher level that requires either an absolute speech recognition ability that corresponds with that of an age-matched SNHL group or meaningful open-set scores for speech in the noisy condition. These two sublevels provide detailed descriptions and classifications of the speech performance of subjects, which is convenient for analysis of the irregular and within-subjects data from AN recipients. The highest objective level of hearing intervention is the achievement of effective social communication, upon which few studies in AN individuals have focused . ImportaAlthough the sample size of the present study was small, the data provide clinical evidence that may serve as a basis for future meta-analyses. A longer period of time may be required to demonstrate the efficacy of CIs in different individuals with AN. Therefore, a long-term follow-up period is needed to demonstrate the dynamic efficacy of CI in prelingual children with AN.Cochlear implantation benefits prelingual children with AN to different extents. The audibility of prelingual children with AN can be reconstructed with implantation, whereas the postoperative speech recognition performance varies among individuals. In the present study, some individuals demonstrated improved speech recognition and others achieved effective speech recognition in the noisy condition. Other subjects did not make progress in speech recognition, and no subject demonstrated effective social communication. Hierarchic assessment can provide a more proper framework by which to determine a subject’s benefits from the CI via a within-subjects and repeated-measures design.


Progressing loss of sensory function is a major problem of aging populations. In humans and animals, loss of auditory function becomes evident by increased hearing thresholds, altered sound processing, and abnormal perception of above-threshold sounds or phantom perceptions like hyperacusis and tinnitus ,2. RecenSynaptic structures, afferent synaptic contacts, and auditory fibers were studied in an animal model on young (3-9 month old) and aged (20-24 month old) rats. The hearing sensation was monitored by auditory evoked brainstem responses (ABR) and otoacoustic emissions (DPOAE) giving insight for the central and peripheral (hair cell function) auditory capacity, respectively. Hyperacusis and tinnitus sensation in the rat were tested using a behavioral approach . We pharAuditory thresholds were increased and the auditory response range was reduced over age. The characteristic changes over age were similar to the changes observed after exposure to traumatizing sound. sGC treatment interacts with the loss of age related and auditory trauma induced loss of hair cell synaptic ribbons in a complex way, proposing that a subclass of auditory fibers with a special vulnerability can be rescued by sGC treatment within a defined time window.Age related and noise induced decline of hearing function are correlated with auditory responses and morphological specifications of hair cell molecular phenotype. The data need to be discussed regarding the proposed cGMP generators in the inner ear and their role for an otoprotective molecular cascade after noise induced damage of the ear or progression of presbycusia.


The most common type of hearing loss results from damage to the cochlea including lost hair cells (HCs) and spiral ganglion neurons (SGNs). In mammals, cochlear HC loss causes irreversible hearing impairment because this type of sensory cell cannot regenerate. The protection from SGN from degeneration has implications for cochlear implant to patients with severe deafness. This review summarizes the several treatments for HC regeneration based on experiments. We discuss how transgene expression of the neurotrophic factor can protect SGN from degeneration and describe potential new therapeutic interventions to reduce hearing loss. We also summarized viral vectors and introduced the gene and drug delivery system for regeneration and protection of cochlear HCs. Finally, we introduce the novel endoscopy we developed for local injection into cochlea. Drug, gene and cell therapy are attractive to facilitate regeneration of hair cells (HCs) and spiral ganglion neurons (SGNs). For endogenous HC regeneration, it is critical to identify potential stem cells of the inner ear.Atoh 1, that can increase the regenerative properties of the sensory epithelium of the cochlea  after HC loss has been shown to reduce the irreversible damage of SGNs .Neurotrophic factors and maintenance of synaptic connectivity are important to prevent neuronal degeneration . Brain-dTreatment with neurotrophic factor such as GDNF significantly enhance SGN survival compared to that in untreated deafened ears. Combining ES with GDNF overexpression treatment additionally protects against SGN degeneration . It suggThe inner ear is enclosed by a bony capsule. Such a relative anatomical isolation makes inner ear an ideal target for therapeutic local injection because spread of the injected vector, the drug, and the transgene to surrounding tissues is expected to be limited.We have several approaches into inner ear including via scala tympani, scala media, or semicircular canal. Scala media approach would be more effective with cells in organ of Corti, but more traumatic. It means via scala media approach does not preserve hearing threshold. Scala tympani approach is less traumatic and used for prevention of SGN degeneration.Gene delivery or transfer introduces exogenous genes into cells. As mentioned above, viral vectors are still the efficient vehicles for gene transfer in animal experiments about the inner ear regeneration.For inner ear gene transfer, several types of viral vectors such as ADV, herpes viral (HSV), adeno-associate viral (AAV) , Sendai in vivo  in cochlea, but not systemic injections due to systemic side effects. However, pharmacokinetics of agents locally injected into inner ear is not well known. Hence, we develop the real time observation of drug delivery system in transgenic animals in vivo . We obseWe also find that RW niche obstruction such as false membrane or fibrous connective membrane blocked drug delivery into inner ear. Therefore, observing RW is very important before local injection of drug into inner ear .The RWM cannot be visualized in around 30% of all the patients with hearing loss. In those cases adhesions need to be removed first before local injection is performed. We develop a novel endoscopy to explore the RWM for the purpose of local injection . The endThe molecular signals that stimulate HCs regeneration and protection of SGNs have begun to be identified. Proliferation of HCs has been achieved in mammals by overexpressing Atoh1, and Notch inhibitor drugs. The preservation of SGNs is very important and has implications for cochlear implants. These studies will lead to therapeutic interventions for the hearing impaired.This manuscript was supported by funds from Grant in aid (OK 2012–2013) for exploratory research in the Ministry of Education, Culture, Sports, Science, and Technology and grant in aid from the Takeda Science Foundation.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Advances in plant science research will lie at the center of efforts to address global food security as demand for agricultural production is predicted to more than double by 2050. A key determinant of plant productivity and thus crop yield is efficient nutrient transport between sites of net nutrient synthesis and acquisition to sites of net utilization. Transfer cells (TCs) play key roles in optimizing such nutrient transport processes in plants, and thus research on these anatomically specialized cell types has the potential to contribute new approaches for improving plant performance. The collection of reviews and research articles in this Research Topic on Transfer Cells provides a valuable contribution to advancing our understanding of these important cell types in plant biology.trans-differentiate from existing cell types by developing extensive wall ingrowths. The resulting increase in plasma membrane surface area enables increased densities of membrane transporters to optimize nutrient transport across apoplasmic/symplasmic boundaries at sites where TCs form. Key challenges in TC biology include understanding the signals and genetic pathways required for initiating and building the unique wall ingrowths that define TC identity. This knowledge in turn will provide a basis to investigate physiological and genetic factors explaining the variability observed in TC differentiation, even among equivalent anatomical situations in highly related species.TCs BETL9 and BETL9like, two lipid transfer genes similar to END-1 expressed specifically in barley endosperm TCs. While the functions of these genes remain unknown, BETL9 is expressed exclusively in the basal endosperm transfer layer (BETL), but protein product accumulates in maternal tissue adjacent to this layer. In contrast, BETL9like is expressed specifically in the endosperm aleurone layer. The expression domains of these genes completely surround the filial tissues, suggesting a protective role at the maternal-filial interface. The miniature1 (mn1) mutant in maize is defective in a BETL-specific cell wall invertase (INCW2), resulting in reduced hexose levels and wall ingrowth development in this tissue. Silva-Sanchez et al.  and GSL11 were induced specifically at low temperature in vte2, only disruption of GSL5 substantially reduced this phloem parenchyma TC-specific production of callose, but had no effect on the low temperature photoassimilate export phenotype of vte2. Adams et al. (Senecio) and companion TCs (pea and Senecio), consistent with the role of these TCs in supporting photoassimilate export from leaves. In contrast, responses to stress such as application of methyl jasmonate caused increased wall ingrowth deposition in phloem parenchyma TCs alone, indicating cell specific responses of TCs to different signals and possibly different roles for these TCs .Arabidopsis provides a powerful genetic system to study TC biology (Arun Chinnappa et al., s et al.  report aCollectively, the insights into TCs provided in the reviews and research articles assembled for this Research Topic establish a valuable platform for continued investigation of these fascinating cell types in plants. Further research on TCs may unlock key possibilities for improving crop yield and thus contribute to the rapidly approaching challenge of addressing global food security.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.


Triclosan (TCS) is widely used as an antibacterial agent in consumer products such as hand soap and toothpaste, and human exposure is widespread. TCS is suspected of having endocrine-disrupting properties, but few human studies have examined the developmental effects of prenatal TCS exposure.We prospectively examined associations between prenatal TCS exposure and anthropometric measures at birth and anogenital distance (AGD) at 3 months of age.Pregnant women from the Odense Child Cohort (n = 514) provided urine samples at approximately gestational week 28 , and urinary TCS concentration was measured by isotope dilution TurboFlow–liquid chromatography–tandem mass spectrometry. Multiple linear regression analysis was used to examine associations between prenatal TCS exposure and measures of size at birth  and AGD at 3 months of age , controlling for potential confounders.Newborn boys in the highest quartile of prenatal TCS exposure had a 0.7-cm  smaller head circumference than boys in the lowest quartile. Additionally in boys, inverse associations of borderline statistical significance were observed between prenatal TCS exposure and abdominal circumference at birth and AGD at 3 months of age . Prenatal TCS exposure was not significantly associated with any of the outcomes in girls. However, AGD was measured in fewer girls, and we observed no significant interactions between a child’s sex and prenatal TCS exposure in anthropometric measures at birth.Prenatal TCS exposure was associated with reduced head and abdominal circumference at birth and with reduced AGD at 3 months of age in boys, although the last two findings were statistically nonsignificant. These findings require replication but are compatible with an anti-androgenic effect of prenatal TCS exposure on fetal growth in boys.Lassen TH, Frederiksen H, Kyhl HB, Swan SH, Main KM, Andersson AM, Lind DV, Husby S, Wohlfahrt-Veje C, Skakkebæk NE, Jensen TK. 2016. Prenatal triclosan exposure and anthropometric measures including anogenital distance in Danish infants. Environ Health Perspect 124:1261–1268; http://dx.doi.org/10.1289/ehp.1409637 Evidence action . Human ee action . Moreovee action . Fetal le action . Howevere action , 2014. Ae action . In a Fre action .Anogenital distance (AGD), the distance from the anus to the genitals, is sexually dimorphic, with males having a 50–100% longer AGD than females . In rodeBecause very little is known about the potential effects of prenatal TCS exposure in humans, the aim of our study was to examine the associations between maternal urinary excretion of TCS as a measure of prenatal TCS exposure and birth outcomes as well as measurement of AGD at 3 months of age stratified by child sex.The study was based on data from the Odense Child Cohort . Brieflyn = 565). The subset of women was selected based on the availability of urine samples. The first 196 samples were selected randomly from among the women enrolled in the Odense Child Cohort between September 2010 and June 2011, whereas the last 369 samples were selected from the remaining women who were enrolled by January 2012 who had available information from questionnaires, urine samples, birth records, and clinical examination of the child at 3 months of age. Of the 565 women, 51 women were excluded because of non-Caucasian origin (n = 30), missing information on ethnicity (n = 16), or missing data on child sex (n = 5), leaving 514 mother-child pairs  eligible for analyses.While recruitment to the Odense Cohort was ongoing, TCS measurements were obtained from urine samples collected at approximately week 28 of pregnancy  from a subset of women with singleton pregnancies  at birth, and birth measures such as birth weight (grams), length (centimeters), head circumference (centimeters) and abdominal circumference (centimeters).Three months after the expected date of birth, regardless of the actual gestational age at birth, the children were invited to a clinical examination , which included measurements of length, weight, and AGD. In addition, genital malformations were noted. Two different measures of AGD were made using a Vernier caliper (SPI DigiMax) in both boys and girls: In girls, a short AGD was measured from the center of the anus to the posterior fourchette (AGDaf), and a long AGD was measured from the center of the anus to the top of the clitoris (AGDac). Correspondingly, in boys, a short AGD was measured from the center of the anus to the posterior base of the scrotum (AGDas), and a long AGD was measured from the center of the anus to the cephalad insertion of the penis (AGDap). Penile width was also measured using a Vernier caliper. In each child, the genital measures were repeated three times, and an arithmetic mean was calculated.n = 245 (AGDas), n = 236 (AGDap), n = 241 (penile width), n = 178 (AGDaf), and n = 176 (AGDac) were included in the statistical analyses.Among the 514 mother–child pairs, 252 males and 179 females had a minimum of one AGD measurement, and 250 males had a minimum of one penile measurement at the clinical examination at approximately 3 months of age. AGD measurements were initiated in August 2011, but owing to technical difficulties in measuring AGD in girls, these data were valid from October 2011. Therefore, data on AGD were available for fewer girls than boys. Because children with only one or two repeated genital measures or who had missing data on the included covariates were excluded, n = 1 (AGDac), n = 1 (AGDas), n = 6 (AGDap), n = 3 (penile width)]. For those with two AGD measurements, the average was used as the outcome measure.Four technicians measured AGD in both the boys and the girls. The coefficient of variation (CV) was < 10% for all the triplicate AGD measurements, except for AGDaf, in which two girls had CVs of 0.10 and 0.14. We conducted subanalyses from which those two girls were excluded. Additionally, we conducted subanalyses in which we also included children who had only one or two AGD measurements  and the samples below the LOD were divided into sex-specific quartiles based on the distributions among the 273 males and among the 241 females. The osmolality-adjusted TCS concentrations and the samples below the LOD were also entered in the statistical model as a continuous variable and were log2-transformed because of skewed distribution. The birth outcomes , the AGD measurements, and the penile width were left untransformed because of acceptable normal distributions of the residuals.TCS concentrations were adjusted for urinary osmolality normalized to the median osmolality of all samples (0.64 Osm/kg) to correct for dilution of the urine. This adjustment was performed for all samples with a measured TCS concentration above the LOD by dividing the individual urinary TCS concentration (nanograms/milliliter) with the individual osmolality of the urine sample (osmoles/kilogram) and multiplying with the median osmolality of all samples (osmoles/kilogram) . Urinarya priori to be important predictors of birth outcomes or AGD. AGD measurements vary with the age and weight of the child, and because the clinical examination was scheduled to take place 3 months after the expected date of birth, we constructed a measure of “post-conceptional age,” which we defined as the sum of the gestational age at birth (days) and the age of the child at the AGD measurements (days). Analyses of associations between TCS and AGD were thus adjusted for the post-conceptional age and the individual weight-for-age standard deviation score (SD-score)  between the genital measures among the boys and among the girls. Differences in the distributions of the TCS concentrations according to population characteristics were assessed by one-way analysis of variance. Multiple linear regression analysis was used to analyze the associations between urinary TCS excretion and birth outcomes and AGD measurements adjusted for potential confounders. We tested for linear trends across TCS quartiles in regression models by means of ordinal TCS quartiles using integer values from 1 to 4. Confounders included in multivariable models were factors known D-score)  calculatD-score) . Additio2) and gestational age (days). All results are presented stratified by sex given a priori interest in sex differences. However, we also tested for potential interaction between child sex and TCS exposure in models with birth weight, birth length, head circumference, and abdominal circumference as outcomes by inserting a product interaction term of child sex × maternal urinary TCS levels  in the statistical models. Percentage change in outcomes was calculated as the difference between the highest exposed group and the reference group divided by the intercept × 100, where the reference values for the covariates were gestational age at birth of 280 days, prepregnancy BMI between 20 and 25 kg/m2, primiparous, and nonsmokers.Analyses of associations between TCS and infant size at birth  were adjusted for parity (primiparous/multiparous), maternal smoking during pregnancy (yes/no), prepregnancy BMI . Associations were considered statistically significant at the All data analysis was performed using SAS v.9.1 (SAS Institute Inc.).The mean age at birth among the 514 women was 31.0 years, 55% of the women were primiparous, and 3.6% smoked during pregnancy. The mean gestational age at birth was 280 days (range 205–296 days) for girls and 280 days for boys (range 221–297 days). Detectable maternal urinary TCS concentration was found in 83% of the samples . The median unadjusted urinary TCS concentration was 0.88 ng/mL, and the 95th percentile and the maximum unadjusted TCS concentration were 428 ng/mL and 2,614 ng/mL, respectively. The median  osmolality-adjusted TCS concentrations stratified by child sex are shown in r = 0.63 (p < 0.0001), and r = 0.61 (p < 0.0001), respectively. As expected, the mean AGD measures in the boys were nearly twice the corresponding AGD measures in the girls  and AGDap .Mean AGD measures and birth outcomes stratified by child sex are shown in he girls . Penile p-trend = 0.01); for every doubling in TCS concentration, head circumference decreased by 0.06 cm among the boys  (p = 0.01) smaller head circumference than boys in the first quartile. This difference corresponds to a reduction of 2.0%  between the fourth versus the first exposure quartile. Maternal urinary TCS level was also inversely associated with abdominal circumference among the boys in a dose-dependent manner, although not statistically significantly (p-trend = 0.07) (p = 0.07). This difference corresponds to a reduction in mean abdominal circumference of 1.8% . We observed no significant associations between prenatal TCS exposure and birth weight , and there was no consistent trend in estimated associations with increasing exposure. We observed a significant positive association between birth length and the third versus the first quartile of TCS, but in the other quartiles, the β values were close to null and without a consistent pattern according to prenatal TCS exposure  . Boys inexposure .We found no significant associations or consistent dose–response relationships between prenatal TCS exposure and any of the measures of size at birth in girls .The interaction terms between child sex and prenatal TCS exposure in models with birth weight, birth length, head circumference, and abdominal circumference as outcomes were not statistically significant , but thep-values < 0.10): every doubling in TCS concentration was associated with a decrease of 0.2 mm  in AGDas and a decrease of 0.2 mm  in AGDap  .Subanalyses showed that inclusion of children with only one or two of the three AGD measurements did not appreciably change the results ; nor did inclusion of examiner information, inclusion of non-Caucasian women, or inclusion of maternal height and prepregnancy weight instead of prepregnancy BMI in the analyses substantially change the observed associations (results not shown).p < 0.10). In contrast, we observed no significant associations between prenatal TCS exposure and any of the outcomes in girls. However, AGD was measured in fewer girls. Analyses of interactions between sex and prenatal TCS exposure were not statistically significant, which was likely because of small sample size and therefore low statistical power to perform interaction analyses.In this prospective study, we observed an inverse association between prenatal TCS exposure as measured by maternal urinary TCS excretion and head circumference in newborn boys. There were no clear associations between prenatal TCS exposure and birth weight or birth length in boys. We also observed reduced abdominal circumference and shorter AGD in boys born to mothers with higher urinary TCS levels, although these estimates did not reach statistical significance (The observed reduction of 0.7 cm in head circumference in boys between the highest versus the lowest exposed quartile was of similar magnitude to the differences in head circumference between boys and girls that were observed in this study (mean difference = 0.6 cm) as well as having been reported at birth in other studies . For exaFew human studies have examined prenatal TCS exposure and birth outcomes, and to our knowledge, no human study has examined associations with AGD. Consistent with the findings of the present study, a French mother–child cohort study including 520 newborn boys observed a statistically nonsignificant inverse association between prenatal triclosan exposure and head circumference, but no associations with length or weight at birth were observed . In a coin vitro studies have suggested different modes of action for TCS, including estrogenic, androgenic, anti-androgenic and gestagenic effects , and because the difference in mean head circumference between boys with the highest versus the lowest TCS exposure was comparable to the average difference in head circumference between boys and girls reported for other populations , we specin vitro studies showing anti-androgenic action of TCS  , suggestn of TCS , althougffspring , and no ffspring .r = 0.30) and girls (r = 0.26).TCS is quickly metabolized with a urinary excretion half-life of < 24 hr . A singlIn this study, a relatively large number of statistical analyses were performed, and some of our results may be chance findings resulting from multiple testing. We had a relatively limited sample size and, hence, limited statistical power for analyses of sex-specific associations between TCS exposure and growth measures. The suggested sex-specific differences in outcomes are therefore hypothetical and should be confirmed in other studies.It is possible that selection bias affected the results. Only 43% of the eligible women participated, and participants were more often of Danish origin and were better educated than nonparticipants. The women included in this study had a lower prevalence of smoking during pregnancy (~4%) than nonparticipants (12%), who were women giving birth between 2010 and 2013 and living in the recruitment area of the Odense Child Cohort . The meap < 0.10). No associations between prenatal TCS exposure and birth outcomes and AGD were found in girls, although AGD was measured for fewer girls and the interactions between child sex and TCS were not statistically significant. Because of the suggestive sex differences, we speculate that our findings are compatible with an anti-androgenic effect of prenatal TCS exposure on fetal development in boys. However, our findings need to be confirmed in other populations, and the biological mechanisms should be elucidated.Prenatal TCS exposure was associated with reduced head circumference and abdominal circumference at birth and with a reduced AGD at 3 months of age in boys, although the last two findings did not reach statistical significance (


To the Editor: Hearing and vision impairments are common in older age. Evidence suggests that these sensory impairments are associated with incident cardiovascular disease (CVD) , stroke),Community‐dwelling men aged 63 to 85  were followed for 10 years, until 2013.During the 10‐year follow‐up, 1,463 deaths occurred, including 408 CVD deaths. In 3,466 men free of prevalent CVD, 489 CVD events, 288 MIs, and 216 strokes occurred during follow‐up. In age‐adjusted analyses, men who could not hear and did not use a hearing aid had greater risks of incident CVD, incident stroke, and CVD mortality compared to men who could hear (Table Men who could not hear and did not use a hearing aid had greater risks of incident CVD, particularly incident stroke, and CVD mortality than men who could hear. Previous research suggests that the associations between hearing impairment and CVD could be attributed to smoking and atherosclerosis,The lack of association between vision impairment and incident CVD and CVD mortality could be due to the definition of vision impairment used, which may have identified severe vision impairment only, thus underestimating the true prevalence of vision impairment. Similarly, dual sensory impairment was not associated with CVD incidence or mortality, which could be due to the small number of men with dual sensory impairment.Hearing impairment in older men was associated with greater risks of incident stroke and CVD mortality. Early detection of hearing impairment in older adults could help prevent CVD. Further research is warranted into the possible mechanisms underlying these associations.


Lawsonia intracellularis (L. intracellularis) is a major concern to the pig industry worldwide. Although 8.3 billion pigs are produced each year in China, few reports on the prevalence of L.intracellularis infection are available. The aim of the current study was to estimate the seroprevalence of L. intracellularis antibodies in intensive pig farms in China.Porcine proliferative enteropathy caused by L. intracellularis were detected using a specific blocking ELISA. Of the 1060 serum samples, 602 were identified as positive using the ELISA test. The apparent seroprevalence of L. intracellularis seropositivity was 57% (95% CI 50 to 64%). The true prevalence  was 77% (95% CI 70 to 83%).A total of 1060 serum samples were collected from 14 commercial pig farms located throughout China. Animals from all age groups were sampled including pre-weaning piglets, weaners, fattening pigs, adult sows and boars. Antibodies against L. intracellularis seropositivity in local breed pigs was significantly less than that in imported breeds. A higher seroprevalence was found in pigs in herds in Central and Northern China, which may correspond to the greater use of the intensive production systems in these areas. We conclude that L. intracellularis is widely prevalent in commercial pigs in China.The highest true prevalence was observed in sows and boars, suggesting that within a herd these stock classes are a reservoir for infection. The prevalence of  Lawsonia intracellularis (L. intracellularis) is a gram-negative, fastidious obligate intracellular bacterium. The pathogen mostly occupies the small and large intestine in pigs and causes porcine proliferative enteropathy (PPE) post weaning. Infection leads to diarrhea, retarded growth and/or sudden death in fattening pigs. A high prevalence of L. intracellularis has been reported throughout the world, contributing to a substantial level of economic loss in the swine industry [industry -3. In soindustry .L. intracellularis infection in pigs was described in 1931 [L. intracellularis has been reported in swine producing countries all over the world. In Denmark, 94% of tested animals were positive by PCR on feces [L. intracellularis has been estimated to cost the industry in the order of USD 25 per sow annually [The first case of  in 1931  and sincon feces , in Swedon feces  and in Kon feces  47% of hon feces  and 84% on feces . In Austannually  and direannually .L. intracellularis infection receives comparatively little attention from animal health authorities compared to highly pathogenic infections such as porcine reproductive and respiratory syndrome (PRRS), classical swine fever (CSF) and Streptococcus suis infection.Highly intensive management of domestic pigs is widely promoted in mainland China, where there is an estimated pig population of approximately 8.3 billion . Severe L. intracellularis was identified from the intestinal mucosa of infected pigs in Southern China [L. intracellularis infected pigs identified by a PCR method was 14% and 16%, in weaners and finishers, respectively in Guangxi province, Southern China [L. intracellularis infection in pigs in other areas of China has not been reported. Moreover, the major transmission routes are unclear in different stock classes and production systems. In this study our aim was to determine the seroprevalence of L. intracellularis in pigs raised in the major pig-producing provinces in China. An additional aim of our study was to document the association between L. intracellularis positivity and the presence of diarrhea.In 2008, the first isolate of rn China . The apprn China . To the L. intracellularis infection in Chinese pigs. Seven provinces  took part in the study. Sampling was carried out using a two-stage cluster design. Two herds from each of the seven provinces were selected at random from a sampling frame of 55 intensive pig cooperatives listed by the Chinese Ministry of Agriculture. Sample size calculations were carried out to determine the appropriate number of individual pigs to be sampled from each of the selected herds. Previous reports have estimated the within-herd prevalence of L. intracellulari infection to be around 13% in weaning pigs and 16% in finishing pigs [L. intracellularis infection was 0.06. We assumed an average cluster (i.e. herd) size of 80 pigs. Sample size calculations were carried out on the basis that we wanted to be 95% certain that our final estimate of L. intracellularis prevalence was within 5% of the true population value.A cross-sectional survey carried out between January and May 2011 to estimate the seroprevalence of ing pigs . Based oA total of 1060 serum samples were collected from pigs from the 14 herds that took part in the study. This included 147 sera from pre-weaning piglets (2 to 4 weeks of age), 221 sera from weaned piglets (5 to 7 weeks), 279 sera from fattening pigs (8 to 14 weeks), 255 sera from adult sows, and 158 sera from boars. Landrace and Large White (breeds exotic to China) and domestic breeds (Meishan and Jinhua) were represented in the sample. The pig numbers were 332, 287, 211 and 230 for Landrace, Large White, Meishan and Jinhua pigs, respectively. Serum samples were stored at −80°C until assayed.L. intracellularis seropositivity and the presence of diarrhea, 659 sera were obtained from herds with animals having a recent history of clinical diarrhea while an additional 401 samples were collected from herds without a recent history of diarrhea.To identify the association between L. intracellularis using a blocking ELISA purchased from Synbiotic Europe SAS  according to the manufacturer’s instructions. The sensitivity and specificity of this test was 72% and 93%, respectively [All sera were tested for antibodies against ectively .L. intracellularis seropositivity was calculated using the number of pigs identified as seropositive divided by the total number of pigs tested. Apparent prevalence estimates were then converted to true prevalence estimates using the approach proposed by Rogan and Gladen [L. intracellularis infection and the average cluster size. The significance of differences between the true prevalence L. intracellularis seropositivity for pigs in different groups  was assessed by considering their confidence limits. If the confidence intervals for two groups did not overlap, the inference was that the two prevalence estimates were significantly different at the alpha level of 0.05 [The apparent prevalence of d Gladen . The Rogd Gladen ,20. Conf of 0.05 . All the of 0.05 .This study was approved by the Ethical Committee for Animal Experiments at China Agricultural University, Beijing, China.L. intracellularis antibodies were found in pigs of all ages. The true prevalence of L. intracellularis seropositivity was 77% (95% CI 70 to 83%). The true prevalence of L. intracellularis seropositivity ranged from 68-87% on stock class.With respect to provinces, true prevalence of seropositivity varied, ranging from 43% (95% CI 22 to 64%) in Guanxi, Southern China to 100% (95% CI 100 to 100%) in Hubei, Central China. Overall, the true prevalence of seropositivity was significantly greater in Central China compared with Southern China and significantly greater in Central China compared with Northern China  and Large White  pigs compared with the local Meishan  and Jinhua  breeds . A higher rate of seroprevalence was found in fattening pigs, sows and boars compared with pre-weaning piglets and weaners. Breeds exotic to China had a higher seroprevalence compared with domestic pig breeds. This is the first report on the infection of L. intracellularis across China. The above evidence indicates that L. intracellularis infection is widespread among intensive pig farms in China.In this study, using a blocking ELISA, the overall true prevalence of L. intracellularis attenuated vaccine, but a high seroprevalence was found in the collected serum samples indicating exposure to the bacterium. The apparent seroprevalence estimates in this study are comparable with those reported in other studies. Apparent seroprevalence was 90% for sows and 56% for fattening pigs in a study conducted in the USA [L.intracellularis-specific antibodies and the mean within-herd prevalence of positive samples was 84% [L.intracellularis contributes to the different serological prevalence estimates reported in Europe and Asia [In this study, none of the pigs had been immunized with  the USA , while g was 84% . Variatiand Asia ,21.L. intracellularis seropositivity was similar for sows and boars and markedly higher than that reported for similar stock classes in other countries. Adult sows with active infection are likely to transmit infection to suckling piglets and infected boars transmit pathogens to sows by artificial insemination. Based on a single assessment of a herd’s diarrhea status we note that the prevalence of seropositivity in herds with a recent history of clinical diarrhea was similar to that of herds without a recent history of diarrhea which would imply some latency of L. intracellularis in sows and boars. The high true prevalence of seropositivity in pre-weaning piglets and boars free of diarrhea does not exclude the possibility that diarrhea was caused by other pathogens. Pig diarrhea is caused by a number of pathogens and mixed infections, including E. coli, Brachyspira hyodysenteriae, Lawsonia intracellularis, and Salmonella spp. [Pigs reared in herds in Central China had true prevalence estimates that were higher compared with pigs reared in herds in the North and South of the country. We speculate that the reason for this finding is that higher seroprevalence rates are associated with intensive pig production units which are common in Central China. The apparent prevalence of lla spp. .L. intracellularis seropositivity identified in this study can be explained by a number of factors. First, inappropriate use of in-feed antibiotics contributes to L. intracellularis infection. In some situations there is a risk of animals developing a resistance to antibiotics such as tetracyclines, sulfonamides, fluroquinalones and zinc bacitracin that are routinely used in feed formulations [L. intracellularis seropositivity is known to be associated with withdrawal of in-feed medication in fattening pig diets. In this study, the true prevalence of L. intracellularis seropositivity for fattening pigs  was significantly greater compared with pre-weaning piglets  and weaners . To some extent, a low true prevalence of L. intracellularis seropositivity reflects the presence of long-term treatment with antibiotics [The high true prevalence of ulations . A recenulations  indicatiL. intracellularis seropositivity is that the presence of infection in breeding herds may arise from the introduction of breeding pigs into a herd without application of appropriate quarantine measures. Every year, thousands of European and American breeding pigs are purchased and imported into China by commercial pig farmers. In most situations the risk awareness of the introducing L. intracellularis infection is underestimated and clinical cases of L. intracellularis infection are often misdiagnosed by local veterinary authorities as well as the pig purchaser. The apparent prevalence of L. intracellularis infection in exotic breeding pigs ranged from 26% to 74% in previous reports [L. intracellularis is spread between herds mainly through the purchase of infected pigs and replacement stock [A second explanation for the high true prevalence of  reports . One sur reports . Recent nt stock ,28. The nt stock .L. intracellularis both within and between herds. In this study, the true prevalence in boars could have contributed to infection rates in sows bred by artificial insemination. In the herds that took part in this study, sows were mated using boar semen without antibiotic treatment. The impact of this practice on between-herd spread of infection needs to be clarified.In addition to the above, transmission from boars is likely to play a role in the spread of L. intracellularis seropositivity is relatively high in Chinese pigs. The highest true prevalence was observed in sows and boars, suggesting that within a herd these stock classes are a reservoir for infection. The prevalence of L. intracellularis seropositivity in domestic pigs was significantly less than that in imported breeds. A higher seroprevalence was found in pigs in herds in Central and Northern China, which may correspond to the greater use of the intensive production systems in these areas.We conclude that the prevalence of This work was supported in part by NSFC grant 31172305 and Ministry of Science and Technology (MoST) grant 2012AA101302-4 (Cheng He). The authors declare that they have no competing interests.ZW, YL and QP contributed to the study design, evaluated the data, and drafted the manuscript. DT was responsible for the coordination of the work and collected blood samples. PH contributed to the revision and CH contributed to the study design, obtained the funding, and corrected manuscript. All authors have read and approved the final manuscript.


Cochlear fibrocytes in the lateral wall region play a critical role in the regulation of inner ear ion and fluid homeostasis, although these are non-sensory cells. Along with other non-sensory cells, fibrocytes in the spiral ligament have been reported to repopulate themselves after damage. However, the studies of regeneration of cochlear fibrocytes have been difficult because a suitable fibrocyte-specific degeneration model did not exist. Therefore, we analyzed cochlear fibrocytes using a rat model of acute cochlear energy failure induced by a mitochondrial toxin. This model is unique because hearing loss is caused by apoptosis of fibrocytes in the cochlear lateral wall not by damage to sensory cells. Although this model involves severe damage to the cochlear lateral wall, delayed spontaneous regeneration occurs without any treatment. Moreover, partial hearing recovery is accompanied by morphological remodeling of the cochlear lateral wall. Two hypotheses are conceivable regarding this spontaneous recovery of cochlear fibrocytes. One is that residual cochlear fibrocytes proliferate spontaneously, followed by remodeling of the functional region of the lateral wall. Another is that some foreign cells such as bone marrow-derived cells promote morphological and functional recovery of the lateral wall. Acceleration of the lateral wall recovery promoted by these mechanisms may be a new therapeutic strategy against hearing loss. Hearing loss is one of the most common disabilities in the world, particularly in aged populations, and its prevalence is increasing. In the United States, 35% of individuals between 65 and 79 years old report hearing impairment; among those aged ≥80, the corresponding figure is 53% . Moreove+/K+-ATPase and Na+/K+/Cl- cotransporters. These molecules are essential for ionic homeostasis and maintenance of the endocochlear potential . This type of delayed recovery of hearing is occasionally observed during treatment of sudden deafness in the clinic (A recent study revealed that cochlear fibrocytes in the lateral wall can repopulate themselves after severe 3-NP-induced degenerative changes accompanied with profound hearing loss . This coe clinic .Hearing recovery in the late phase in patients with sudden deafness is rarely detected, even if it occurs. The mechanism of clinical recovery of hearing remains unknown, but clinical course of the delayed recovery of hearing is similar to that involved in 3-NP-induced hearing loss. At least the spontaneous regeneration of hair cells or spiral ganglion cells never occurs because a loss of sensory cells is irreversible in mammals once these cells are gone . TherefoSeveral mechanisms of lateral wall regeneration have been proposed. One is local proliferation of the fibrocytes that can survive the damage. It is reported that cochlear fibrocytes can continue to divide, although the proliferation capacity is reduced when the animal is at an advanced age . MoreoveAn additional hypothesis is that some substance that migrates into the cochlear lateral wall accelerates the regeneration of fibrocytes. Several studies demonstrated that the spiral ligament contains bone marrow-derived cells that can differentiate into macrophages in a deafened cochlea after acoustic trauma . Other rTherefore, acceleration of fibrocyte repopulation is believed to be a promising therapeutic strategy for treating some types of sensorineural hearing loss. Further research is expected to reveal the detailed mechanism behind the hearing recovery via fibrocyte regeneration.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


SLC26A4 and the mouse ortholog Slc26a4 code for the protein pendrin, which is an anion exchanger expressed in apical membranes of selected epithelia. In the inner ear, pendrin is expressed in the cochlea, the vestibular labyrinth and the endolymphatic sac. Loss-of-function and hypo-functional mutations cause an enlargement of the vestibular aqueduct (EVA) and sensorineural hearing loss. The relatively high prevalence of SLC26A4 mutations provides a strong imperative to develop rational interventions that delay, ameliorate or prevent pendrin-associated loss of cochlear and vestibular function. This review summarizes recent studies in mouse models that have been developed to delineate the role of pendrin in the physiology of hearing and balance and that have brought forward the concept that a temporally and spatially limited therapy may be sufficient to secure a life-time of normal hearing in children bearing mutations of SLC26A4.The human gene  SLC26A4 (MIM #605646) codes for the protein pendrin, which is an electroneutral exchanger for anions such as HCO3−, Cl−, and I− , which contains a response-element for the doxycycline-bound transactivator to mediate the expression of murine Slc26a4. Both transgenes were crossed into the Slc26a4Δ/Δ line. In the presence of doxycycline, pendrin was expressed in the inner ear at natural times and natural sites. Omission or withdrawal of doxycycline prohibited expression or led to the rapid cessation of pendrin expression. Thus, pendrin expression could be controlled through doxycycline. Interestingly, loss of pendrin expression in a fully functional inner ear did not affect hearing , Tg[R]nner ear . This mo hearing . This fiSlc26a4Δ/Δ was used to determine the time period during which pendrin expression is required for normal cochlear development. The critical time period during which pendrin is needed for the development of normal hearing was between E16.5 and P2 [The mouse model Tg[E], Tg[R]5 and P2 . The timSLC26A4. The concept that a temporally and spatially limited therapy may be sufficient to restore normal hearing provides an imperative to develop interventions that secure a life-time of normal hearing in children bearing mutations of SLC26A4.Studies in mouse models have provided tremendous insights into the role of pendrin in inner ear development. Moreover, studies of pendrin-related mouse models have revealed pathobiological mechanisms that may have broad implications beyond hearing loss associated with loss-of-function or hypo-functional mutations of