The burden of disease of hearing disorders among adults is high, but a significant part goes undetected. Screening programs in combination with the delivery of hearing aids can alleviate this situation, but the economic attractiveness of such programs is unknown. This study aims to evaluate the population-level costs, effects and cost-effectiveness of alternative delivering hearing aids models in Tamil Nadu, IndiaIn an observational study design, we estimated total costs and effects of two active screening programs in the community in combination with the provision of hearing aids at secondary care level, and the costs and effects of the provision of hearing aids at tertiary care level. Screening and hearing aid delivery costs were estimated on the basis of program records and an empirical assessment of health personnel time input. Household costs for seeking and undergoing hearing health care were collected with a questionnaire . Health effects were estimated on the basis of compliance with the hearing aid, and associated changes in disability, and were expressed in disability-adjusted life years (DALYs) averted.Active screening and provision of hearing aids at the secondary care level costs around Rs.7,000 (US$152) per patient, whereas provision of hearing aids at the tertiary care level costs Rs 5,693 (US$122) per patient. The cost per DALY averted was around RS 42,200 (US$900) at secondary care level and Rs 33,900 (US$720) at tertiary care level. The majority of people did consult other providers before being screened in the community. Costs of food and transport ranged between Rs. 2  and Rs. 39 .Active screening and provision of hearing aids at the secondary care level is slightly more costly than passive screening and fitting of hearing aids at the tertiary care level, but seems also able to reach a higher coverage of hearing aids services. Although crude estimates indicate that both passive and active screening programs can be cautiously considered as cost-effective according to international thresholds, important questions remain regarding the implementation of the latter. The burden of hearing loss among adults is large. According to WHO estimates, some 413 million people suffer from mild or greater hearing loss globally, an estimated 187 million people from moderate or greater hearing loss, and 46 million people from severe or profound hearing loss . WHO estIn this respect, two important observations can be made. Firstly, active screening for hearing disorders has been suggested as an effective strategy to increase case detection, and a number of studies in a range of developing countries have shown its feasibility in combination with provision of hearing aids . HoweverIn response to these two observations, this paper reports on the costs and health effects of two active screening programs in the community in combination with the provision of hearing aids at secondary care level, and the costs and effects of the provision of hearing aids at tertiary care level in three sites in Tamil Nadu, India, in the period 2007–2008. This study is part of a number of WWHearing pilot projects on the delivery of hearing aids in developing countries .The study evaluated three different programs:1. Active screening and fitting at secondary care levelActive screening in 62 communities in Karigiri through hearing camps and fitting of hearing aids to 101 patients by the community hearing workers in the Karigiri secondary level health facility. This program also fitted hearing aids to 111 patients who presented themselves directly at Karigiri secondary level health facility for treatment;2. Active screening and fitting at secondary care levelActive screening in 63 communities in Vellore through hearing camps and fitting of hearing aids to 163 patients by the community hearing workers in Community Health and Development (CHAD), a department of the Christian Medical College, Vellore providing primary and secondary health care. This program also fitted hearing aids to 32 patients who presented themselves directly at CHAD for treatment;3. Passive screening and fitting at tertiary care levelPassive screening and fitting of hearing aids to 100 patients in the ENT department of the Christian Medical College (CMC), a tertiary level health facility in Vellore.In both active screening models (programs 1 and 2), community hearing workers screened adults in the community. A selection of those receive a hearing assessment, for eligible patients an ear mould impression was taken, and the impressions were then sent for conversion into ear moulds at tertiary care level (CMC). Subsequently, at the secondary level health facility, the programmable hearing aid was fitted by the community hearing worker. In both programs 1 and 2, all people received monaural hearing aids. People were referred in cases where disorders were not treatable with hearing aids. The patient was followed-up in a total of four visits within a year  and after that at yearly intervals, also by the community hearing worker. The programs also served patients who present themselves directly for treatment. Community hearing workers attended a three week training program on basic hearing health care, including basic audiometry, ear mould impression taking and hearing aid fitting.In the passive screening mode (program 3), patients presented themselves at the outpatient department of the tertiary level health facility, and were referred to the audiology department in case of hearing problems. The consultation with the audiologist included a physical and a standard audiometric examination. People were referred in cases where disorders were not treatable with hearing aids. For patients eligible for a hearing aid, an ear mould impression was taken by an ear mould technician. At a second visit, a digital, programmable hearing aid was fitted. In program 3, some people received monaural and some binaural hearing aids. The patient was followed-up in a total of four visits within a year  and after that at yearly intervals.In all programs, eligibility of patients for hearing aids was determined with reference to WHO Guidelines for Hearing Aids and Services for Developing Countries . In all The study had an observational design, in which all people were followed during the course of their inclusion in the program. The costing analysis followed WHO guidelines on costing and cost-effectiveness analysis  where poprior to enrolment in the present program.Guidelines on cost-effectiveness analysis (CEA) typically advocate adoption of the societal perspective in the calculation of costs and effects ,8. This We only include health effects and not effects of improved hearing on labor productivity. To our knowledge, no study has assessed the health state valuation of deafness for adults in India, and following WHO guidelines on CEA, we use health state valuations from the Global Burden of Disease study . The heaThe study was performed in the context of the routine service program, and no specific ethical approval was required. All respondents gave informed consent to their participation in this study.Patient volumes and costs for the various programs are summarized in Table Health care costs of the active screening programs included fixed and variable costs  in program 3 to Rs 1,383,120  in program 2. The total costs per person fitted with hearing aids ranged from Rs 5,381 (US$122) in program 3 to around Rs. 7,000 (US$152) in programs 1 and 2  and Rs. 53  between the different visits and sites. Costs of food and transport ranged between Rs. 2  and Rs. 39 .Sensitivity analysis was applied to test the robustness of results towards the use of alternative values on key parameters, including the proportion of patients actually wearing hearing aids, the lifetime of the hearing aids, the difference in health state valuation between treated and untreated deafness, and cost of hearing aids. This has a varying impact on study results, individually, and in combination  Table . Also, wActive screening for hearing impairment, including the provision of hearing aids to eligible patients at primary or secondary care level, is slightly more costly than fitting of hearing aids to those people who present themselves for treatment at the tertiary care level. Cost differences between the different programs – as cost per patient fitted with hearing aids – are maximally some 25%, and can be, given uncertainty about study assumptions, considered to be moderate only. Active screening programs seem to be better able than passive screening programs to reach a higher coverage of hearing aids services in the community, and whether this is worth the slightly higher costs is a matter of judgment.A number of issues need to be taken into account when considering the costs estimates. First, and most obvious, active screening programs do require significant human and material resources, and these costs add to the average costs of hearing aids fitted. The cost savings stemming from treatment at secondary care level instead of treatment at tertiary care level, do offset these costs to a certain extent. Second, programs 1 and 2 basically concern two components, i.e. an active screening component in the community that refers people to the respective health facility, and another component that involves people who present themselves directly at the health facility for treatment. We analyze the components in combination, as the second component would not exist without the first component being in place .In addition to the detailed cost estimates, this paper also made some crude estimates of related health effects, and cost-effectiveness of the hearing aid delivery models. We estimated that the cost per DALY averted ranged between around US$720 (passive screening) and US$900 (active screening). WHO labels interventions as cost-effective if they cost less than three times GDP per capita, and as not cost-effective if they cost more than three times GDP per capita . India hAn interesting observation is that the majority of patients in the active screening programs had visited a different health provider before being included in the program under study. This indicates that the hearing disorders of the people in the present program did not go undetected prior to the active screening program, but rather that people did not seek care with appropriate health providers. One possible explanation is that hearing aids were not delivered free of charge prior to the present program (as they were in the present program), but at significant costs ), and may not have been affordable to households. This poses a question concerning the relevance of the screening program, and whether the delivery of (highly) subsidized hearing aids, in combination with an (community-based) information campaign publicizing their availability, may not reach the same objective.Our study has a number of limitations. Firstly, we only made a crude assessment of the health effects of hearing aids by using health state valuations derived from the Global Burden of Disease study. This approach does not differentiate between the capacities of the secondary and tertiary health facility levels to diagnose hearing problems and fit hearing aids. However, recent analyses shows that the secondary and tertiary care health facilities as included in our study, are equally effective in that respect . Second,Fourth, if screened people are referred for consultation, but are not found eligible for hearing aids, health facilities provide additional diagnostic tests for and treatment of ear-related health problems as a standard procedure . Related health effects and costs were not included in the analysis, as the decision to carry out these diagnostic tests and treatment stands by itself  and shouBaltussen et al.  recentlyBased on the present study and the study in China , a numbeActive screening and provision of hearing aids at the secondary care level is slightly more costly than passive screening and fitting of hearing aids at the tertiary care level. However, the former carries the benefit that it seems able to reach a higher coverage of hearing aids services and it can be combined with the screening and management of other diseases in the community. Although crude estimates indicate that both passive and active screening programs can be cautiously considered as cost-effective according to international thresholds, important questions remain regarding the implementation of the latter.The authors declare that they have no competing interests.RB, VJA, JA & NG designed the study. VJA, NG, BA, JA, JA, and MP collected the data. RB analyzed the data. All authors contributed to the drafting of the manuscript.The pre-publication history for this paper can be accessed here:Appendix tables. Tables forming appendixClick here for fileAppendix questionnaire. Questionnaire about the costs of hearing problems and its treatment.Click here for file


The aims of this study were: (1) To examine the presence of alphafetoprotein (AFP) bearing tumour cells in PBSC harvests from advanced GCT patients obtained after multiple cycles of induction chemotherapy. (2) To determine whether induction chemotherapy contributed to in vivo purging of the tumour. We evaluated cryopreserved PBSC samples from 5 patients with advanced stage II/III AFP producing GCT. PBSC were separated after the first, second and third cycles of induction chemotherapy. Those samples were analysed using the nested reverse transcription polymerase chain reaction (RT-PCR) method to detect AFP mRNA. Although, in all patients, AFP mRNA was detected in PBSC samples after the first or second cycle of induction chemotherapy, but was not detected in 3 of 4 samples after the third cycle of chemotherapy. Although it is not clear whether tumour cells contaminating PBSC fraction contribute to disease relapse, PBSC harvested after at least 3 cycles of induction chemotherapy might be recommended to avoid such a possibility. © 2001 Cancer Research Campaign


More recently it has been shown that the addition of small amounts of certain excipients to a mucoadhesive formulation can lead to a substantial decrease in observed mucoadhesion in an in vitro test system, which suggests that formulation of these systems could be crucial in developing successful dosage forms. A series of experiments has been carried out which indicates that the presence of flow aids at high concentrations present in tablets can affect the observed ex-vivo mucoadhesive bond. Magnesium stearate (5%) exerts its negative effect on the mucoadhesion of Carbopol® 971P and polyoxyethylene combination by hindering the hydration of the polymer. Adhesion time of formulation containing 5% magnesium stearate was found 4.7±0.34 h and percent hydration of same formulation was 70.12%.Talc and colloidal silicon dioxide (Aerosil), which do not possess the same hydrophobic properties or have available divalent cations were found to be viable alternatives to magnesium stearate.The aim of present study is to investigate the effect of flow aids on the observed  In addiage forms7.® 971P were obtained as a gift sample from Colorcon, India Ltd, Goa. Flow aids like magnesium stearate, talc, aerosil  were obtained as a gift sample from Glenmark Pharmaceuticals Ltd., Sinnar, India. All other reagents used were of analytical grade.Polyoxyethylene and Carbopol® 971P and flow aids were compressed using 16 station rotary tablet compression machine (Rimek) using punch 15×6.5 mm. Hardness of all the disks was maintained in between 65-70 N (Disks (320 mg) of polyoxyethylene, Carbopol 65-70 N .Adhesion time of polymeric discs was determined by using rotating cylinder method USP type VI apparatus  at 37±0.5° at 100 rpm using 0.1N HCl as a medium[Swelling index or % of hydration= ((W2−W1)/W2)×100, where, W1 is the initial weight of tablet, W2 is the weight of disks at 12 h. Swelling study of individual polymers and combinations was carried out using rotating paddle at 100 rpm  and 0.1 N HCl as medium, temperature was maintained at 37±0.5°. The disk was adhered to goat gastric mucosa which was attached to the paddle using cynoacrylate glue and the weights of disks were noted at 1 h intervals (W2).® 971P and polyoxyethylene combination by hindering the hydration of the polymer, consequently causing a decrease in the number of carboxylic acid moieties available for bonding to the mucus. The material may also block the passage of water from mucus to the mucoadhesive, which normally occurs by the mechanism of mucus dehydration. In addition it is known that divalent cations can crosslink polyacrylic acids and this may contribute to the deleterious effects that the high concentration of magnesium stearate has on the observed mucoadhesion. It is also observed that Talc and colloidal silicon dioxide (Aerosil) do not have any significant effect on adhesion time and percent hydration, even at 5% concentration . Accordi


Hearing impairment is most accurately measured by a clinical pure-tone audiogram. This method is not suitable for large-scale, population-based epidemiological studies as it requires that study participants visit a clinic with trained personnel. An alternative approach to measuring hearing ability is self-estimation through questionnaires, but the correlation to clinical audiometric tests varies. To evaluate an Internet-based hearing test pilot compared to a question about self-estimated hearing and the feasibility of using an Internet-based hearing test and an Internet-based questionnaire in a population of 560 members of the Swedish Hunters’ Association in the age group 20-60 years. An invitation was mailed to the participants in March 2007 together with the URL to the study Web site, a personal username, and a password. The Web site included the questionnaire, the hearing test, and instructions for participating in the study. The hearing test resembles a clinical audiogram presenting 6 tones between 500 and 8000 Hz. Tones are presented between 0 and 60 dB, and the participant responds to the tones by pressing the space bar. The hearing test requires headphones and is based on JAVA programming. Before the participant can start the hearing test, it has to be calibrated against a reference person with good hearing between 15 and 35 years of age.P = .003) and the correct version of the JAVA program (P = .007) than those who only answered the questionnaire. After 5 months, 162 out of 560 (29%) had answered the questionnaire, out of which 88 (16%) had completed the hearing test. Those who actively declined participation numbered 230 out of 560 (41%). After removing duplicates and hearing tests calibrated by unreliable reference data, 61 hearing tests remained for analysis. The prevalence of hearing impairment from the Internet-based hearing test was 20% (12 out of 61), compared to 52% (32 out of 61) from the self-estimated question. Those who completed the hearing test were older than the non-participants, and more had headphones ( Though an Internet-based hearing test cannot replace a clinical pure-tone audiogram conducted by a trained audiologist, it is a valid and useful screening tool for hearing ability in a large population carried out at a low cost. Hearing loss is one of the most common physical impairments in the western world and is an increasing problem among younger age groups . The golDigital technologies provide the possibility of developing computer-based programs for measurement of physical impairment. A number of commercial programs resembling clinical audiograms for measuring hearing are available online ,13. VariP-value < .001) for the right ear and 0.93  for the left, and the Internet-based hearing test had a 75% sensitivity and a specificity of 96% compared to the clinical audiogram. The Internet-based hearing test is not a substitute for pure-tone audiometry for diagnosis of hearing loss, but rather should be used to screen for hearing ability in longitudinal, large-scale, population-based studies. Therefore, the sensitivity is sufficient.We have developed an Internet-based hearing test resembling a clinical pure-tone audiogram. The hearing test aims at measuring real-time hearing ability in large-scale epidemiological and clinical studies in the participant’s home environment, using headphones and a home computer. The Internet-based hearing test has been validated against a pure-tone audiogram at the Karolinska University Hospital . Out of This paper evaluates the pilot study testing the feasibility of collecting epidemiological data on hearing ability using an Internet-based hearing test together with an extensive questionnaire including questions about self-estimated hearing prior to the test. The study is also evaluated in terms of willingness to participate and possible reasons for non-participation, including technical obstacles.A pilot study was designed to test the feasibility of conducting a large-scale cohort study among more than 200,000 hunters and marksmen from the Swedish population. The larger study aims at studying the relationship between noise-induced hearing loss, exposure to heavy gun shots, and the use of hearing protection.The participants enter the study through a Web site that includes a Web-based questionnaire and an Internet-based hearing test. To enter the Web page, the participant enters a personal username and password. The participants cannot access the hearing test before filling in the questionnaire. The questionnaire includes 12 sections with, in total, approximately 100 questions regarding background, hunting, self-estimated hearing, occupation, military service, problems with hearing, medications, and recreational activities. The question about self-estimated hearing was stated as, “How is your hearing?”, and the optional answers were “good”, “minor hearing loss”, “moderate hearing loss”, or “severe hearing loss”.The hearing test is based on JAVA 5.0, and before the participants can start the hearing test, they are instructed to verify whether or not the computer has the correct version of the JAVA program. If not, the JAVA program can be downloaded free of charge. Before the participants can start the hearing test, the sound levels are calibrated against a reference person to compensate for variations in different headphones and noise interference from the computer and surroundings. Prior to the calibration test, the participant and the reference person are instructed to follow guidelines on how to set correct volume settings on the computer as well as using the headphones. In the following calibration phase, the reference person enters age (preferably between 14 and 35 years) and gender. The reference person is presented with a volume slider having a fine-tuned scale ranging over 30 dB. The reference person is instructed to move the slide head to a barely audible position, which is the reference hearing level (RefHL) for the frequency, and then request the program to present the next tone. The tone is a frequency-modulated sinus tone—a slightly vibrating tone which can be heard on headphones having “dead points” at certain pure frequencies. This tone is presented to both ears to get the lowest hearing threshold for each ear. The procedure starts from 500 Hz and is repeated for 1000 Hz, 2000 Hz, 4000 Hz, 6000 Hz, and 8000 Hz.Quality check of the calibration of the RefHL data is performed on the finalized data. It is limited to a maximum check of a 15 dB difference over the frequencies, along with a check of whether the reference person has moved the volume slider for each frequency.During the hearing test, intensity levels are presented between 0  and 60 dB sound pressure level (dBSPL). The hearing test starts by presenting the 500 Hz tone to the left ear for 1 second at 30 dBRefHL, which is 30 dB higher than the hearing threshold set by the reference person for that frequency. The tone is followed by a shorter pause of random length to discourage the participant from guessing. The participant presses the space bar on the computer keyboard to register that a tone is heard. The key press is accepted as registering a threshold if the key is pressed within the presented tone timeframe, adjusted for the human reaction time. If the space bar is pressed half a second after the accepted timeframe, it is registered as too late and considered “imagined”. This is not accepted as a threshold measure. When a tone is registered as heard, the test presents the same frequency at a 6 dB lower intensity level. When a tone is not heard, the test instead presents a tone at a 6 dB higher intensity level. The test proceeds for both left and right ears to settle the hearing levels for each frequency. This test procedure is a Web adaptation of established clinical audiometric testing and follows the guidelines for clinical audiometric testing .After completing the test, the participant is shown an audiogram presenting the hearing levels for both ears at each measured frequency.In March 2007, an invitation letter was sent to 560 members of the Swedish Hunters’ Association. Subjects were selected proportionally to the distribution among the members in terms of gender  and age (in the age group 20-60). The mailed invitation included a description of the study and a personal username and password. The invitation also included a prepaid return letter which the participants could use to decline participation. This letter included a voluntary question about their reason for non-participation. The data collection was closed in August 2007. During the study, 2 paper reminders were sent, followed by a telephone reminder. The first reminder was sent 3 weeks after the initial invitation, followed by a second reminder after an additional 3 weeks and a telephone reminder 3 weeks after the second paper reminder. During the telephone reminder, those who declined participation were asked about the reason for their non-participation. Reminders were sent to subjects who had not yet completed the questionnaire and hearing test without declining participation, and to those who had answered the questionnaire but not completed the hearing test.The audiometric data from the hearing test was classified according to the definition by WHO for normal hearing, minor hearing loss, moderate hearing loss, and severe hearing loss . Normal  The study procedure is described with respect to the compliance and dropout at different checkpoints throughout the study . BackgroAfter 3 reminders, 162 out of 560 (29%) had completed the questionnaire (questionnaire respondents), of which 88 (16%) had completed the hearing test (full respondents). After reminders 1 and 2, 146 had actively declined participation, and an additional 84 declined participation during the telephone reminder . There were 154 individuals who could not be reached or did not contact the study center for non-participation, and 14 participants entered the password without completing the study. A flowchart of the participation scheme is presented in                     In total, 126 hearing tests were carried out by 88 unique participants. Among the duplicates, the test with the best  result was used in the analysis. After removal of those hearing tests with an incorrect reference, 61 hearing tests remained for which the mean age was 45 years. Results of the hearing test in comparison to the self-estimated hearing question are shown in P < .001). Only one who had a higher degree of hearing loss had a documented ear injury. Among those who had a hearing impairment according to the Internet-based hearing test, 6 out of 12 (50%) had classified their hearing differently in the self-estimated question. After excluding severe hearing loss, the simple kappa coefficient was calculated to 0.18 , indicating a slight agreement between the two measurements.On the self-estimated hearing question, 32 out of 61 (52%) reported hearing loss; 12 of those 61 (20%) showed hearing loss on the Internet-based hearing test. The Chi-Square test shows this difference to be statistically significant (P = .003) and the correct JAVA version on their computers (P = .007) compared to questionnaire respondents , lack of interest in the study (35%), lack of headphones (13%) (which reflects a difference between questionnaire respondents and full respondents), having no experience of gunshots or hunting, or already experiencing hearing loss and therefore considering themselves to be inappropriate for the study .P < .001). The Internet-based hearing test indicated hearing loss in 20% of the tested individuals, compared to 52% in the self-estimated question. These results could indicate an underestimation of self-estimated hearing ability and display the difficulty of evaluating a self-estimated hearing impairment. The high degree of underestimation could be a result of the difficulties in the calibration procedure of the Internet-based hearing test, resulting in minor hearing loss not being detected. But, as this study population is relatively young (20-60 years), 52% seems to be a high prevalence of hearing impairment, even though the study includes a population with high exposure to impulse noise. In 2005, 14.3% of the Swedish population had a hearing impairment, out of which 63% were still of working age (16-64 years) [This study evaluates an Internet-based hearing test in terms of its agreement to self-estimated hearing assessed by a question in a questionnaire and willingness to participate. Statistically, the results from the hearing test and the self-estimated hearing were significantly different 4 years) . This fi years . 4 years) ,9,10 andThe study also aims at evaluating the willingness to take part in a study including a Web-based questionnaire and an Internet-based hearing test. Our study had a response rate of 29% to the questionnaire and 16% to the hearing test, which is low for an epidemiological study. Full respondents were slightly older than the average non-participant, which might indicate that the older age group had a keener interest in the study. This was expected, as hearing decreases with age.There were no differences between questionnaire respondents and full respondents in terms of sociodemographic characteristics and self-estimated hearing, where the full respondents were a representative sample of the total respondents. The full respondents had, however, greater access to headphones and already possessed the correct version of JAVA prior to the test more often than did the questionnaire respondents. The low response rate might therefore be due to the technique and the many steps prior to the test , rather than personal characteristics. One concern prior to the study was computer and Internet knowledge among the study participants, but Internet use in Sweden is among the highest in the world. In Sweden, 96% of the population can access the Internet from their homes , and an One of the major problems of this study was the calibration and especially the quality of the reference data, as many of the respondents seemed to use a reference person with unreliable hearing. The ability to test the hearing of the reference person is limited. Other Internet-based hearing tests have used a reference tone or a specific program for calibrating the zero level ,15. ThisFor the large-scale study, the calibration technique will be redesigned to measure the reference threshold twice in order to get more reliable values and to better judge that a reference is suitable (hearing loss estimated to be less than 15 dB). Also, the reference person will be asked to answer a couple of questions regarding hunting experience and perceived hearing in order to detect potential bias.Hearing impairment is a growing problem and can occur at all ages. Causes include repetitive exposure to loud sounds, or other external noises . HearingThough the Internet-based hearing test cannot replace an audiogram from a clinical pure-tone audiometer conducted by a trained audiologist, it is a more useful and objective tool for screening hearing in a large population than a self-estimated hearing questionnaire.


The current, continuous National Health and Nutrition Examination Survey (NHANES) has included blood mercury (BHg) and fish/shellfish consumption since it began in 1999. NHANES 1999–2004 data form the basis for these analyses.This study was designed to determine BHg distributions within U.S. Census regions and within coastal and noncoastal areas among women of childbearing age, their association with patterns of fish consumption, and changes from 1999 through 2004.We performed univariate and bivariate analyses to determine the distribution of BHg and fish consumption in the population and to investigate differences by geography, race/ethnicity, and income. We used multivariate analysis (regression) to determine the strongest predictors of BHg among geography, demographic factors, and fish consumption.Elevated BHg occurred more commonly among women of childbearing age living in coastal areas of the United States (approximately one in six women). Regionally, exposures differ across the United States: Northeast > South and West > Midwest. Asian women and women with higher income ate more fish and had higher BHg. Time-trend analyses identified reduced BHg and reduced intake of Hg in the upper percentiles without an overall reduction of fish consumption.BHg is associated with income, ethnicity, residence . From 1999 through 2004, BHg decreased without a concomitant decrease in fish consumption. Data are consistent with a shift over this time period in fish species in women’s diets. Over the past decade, risk assessments describing association between human exposures to MeHg and occurrence of neurologic deficits have been developed by multiple countries and organizations . We discThe current reference dose (RfD) used by the U.S. EPA was based on cord blood measurements and is associated with fetal BHg concentrations of 5.8 μg/L . HoweverPrevious analyses of NHANES BHg data  confirmeNHANES data have been used to describe the distribution of BHg concentrations in a nationally representative sample for adult women  but haveExplaining patterns of MeHg exposure is challenging because MeHg concentrations within and among fish species are known to vary by more than 10-fold . Regionaa) distributions of BHg concentrations and fish consumption that differ across the four U.S. Census regions and between the coastal and noncoastal populations, b) significant temporal trends in BHg levels and fish consumption between 1999 through 2004, and c) BHg concentrations associated with racial/ethnic and income level. Trends in patterns of fish consumption, if present, could be used to assess the impact of national fish advisories and Hg reduction programs. Data on geographic/ethnic groups and socioeconomic associations could be used to target intervention programs. The regional results can be generalized to provide population estimates for the U.S. Census regions.The purposes of the present study of Hg exposures are to provide for the first time separate estimates of BHg distribution and fish consumption for the individual U.S. Census regions and for coastal and noncoastal populations. Such data are useful in determining where to focus efforts to reduce Hg exposure. The present analysis can be used to determine We evaluated data for examinees who participated in NHANES during survey years 1999–2004 to assess the statistical association between seafood consumption and BHg by region of residence, race/ethnicity, and annual income. Further, we examined time trends for both BHg and fish consumption. NHANES is an annual survey conducted by the NCHS. The data include BHg levels, 24-hr dietary recall, and 30-day finfish and shellfish consumption frequency for women 16–49 years of age who reside in the United States. The NHANES sampling frame includes all 50 states. The documentation and publicly available data for NHANES can be found online . The regFollowing NHANES analytic guidelines , we usedIn order to estimate long-term Hg intake, we combined data collected through the 24-hr dietary recall and the 30-day fish frequency questionnaire to estimate 30-day Hg intake  We then defined the four coastal regions based on nearest body of water; for example, counties in California, Oregon, Washington, Alaska, and Hawaii that we defined as coastal were categorized as Pacific Coast. We separated noncoastal counties into four inland regions using the U.S. Census regions; for example, noncoastal counties in California, Oregon, Washington, and Alaska along with the entire states of Idaho, Montana, Wyoming, Colorado, New Mexico, Arizona, Utah, and Nevada became the West region . We also designated the entire state of Florida as coastal, split between the Atlantic Coast and the Gulf of Mexico. We designated Miami-Dade County as Atlantic Coast, and Monroe County as Gulf of Mexico. These subdivisions run the risk of small sample sizes; however, the definition of coastal was sufficiently broad to avoid single primary sampling units.The geographic unit used by NHANES is a county or county equivalent ; therefohttp://www.ehponline.org/members/2008/11674/suppl.pdf).] When we included coastal regions in this analysis, additional spatial heterogeneity in BHg (http://www.ehponline.org/members/2008/11674/suppl.pdf).] Fish species eaten by survey participants varied by region (p < 0.01), Pacific (p < 0.0001), and Gulf (p < 0.0001) coasts had higher BHg concentrations compared with women from the inland West, whereas women from the inland Northeast and inland Midwest had significantly lower BHg levels (p < 0.0001). y in BHg  and estiy in BHg  was appay region , with rey region . BHg incp = 0.07), estimated 30-day Hg intake (p = 0.11), or reported frequency of seafood consumption (p = 0.69). However, in multiple regression modeling, adjusting for covariates including coastal/non-coastal residence, the years 1999–2000 had significantly higher BHg levels (p < 0.0001) compared with 2003–2004, and 2001–2002 had significantly lower BHg levels (p < 0.01) . Although the analyses did not support the conclusion that there was a general downward trend in BHg concentrations over the 6-year study period, there was a decline in the upper percentiles reflecting the most highly exposed women with BHg concentrations greater than established levels of concern . In addition, the percentage of examinees with BHg values ≥ 3.5 μg/L and ≥ 5.8 μg/L was much greater in 1999–2000 compared to 2001–2002 and 2003–2004 (http://www.ehponline.org/members/2008/11674/suppl.pdf)]. The RfD for Hg intake is 0.1 μg Hg/kg body weight (μg/kgbw) per day, or 3.0 μg Hg/kgbw per month (30 days).Analysis of temporal trends through simple regression modeling showed no statistically significant difference among the three sets of study years  for BHg (003–2004 . We foun003–2004 . This suhttp://www.ehponline.org/members/2008/11674/suppl.pdf).] Results also showed that self-selected ethnic identity was associated with total BHg concentrations, estimated 30-day Hg intake, and frequency of either finfish or shellfish consumption. For example, BHg levels, reported frequency of seafood consumption, and 30-day Hg intakes were highest among women who designated themselves as being in the “other” category .  In addition, women from families reporting incomes of ≥ $75,000 (the reference category) had statistically higher BHg levels than did women from families with incomes of ≤ $55,000 (p < 0.01). In all cases, BHg concentrations were also significantly associated with age and estimated 30-day Hg intake (p < 0.0001). We identified statistically significant relationships between higher income and, respectively, increasing BHg concentration (http://www.ehponline.org/members/2008/11674/suppl.pdf)]. Non-Hispanic blacks (p < 0.0001) and women grouped in the “other” racial category (p = 0.002) had significantly higher BHg concentrations than did non-Hispanic whites.In multiple regression modeling, after adjusting for other factors related to BHg, both race/ethnicity and income remained statistically significant predictors of BHg levels observed in this study  , BHg conA more complex association between income and racial/ethnic group may also exist. According to the 1990 U.S. Census , the medOur analysis of 30-day Hg intake indicated that there was no consistent trend in fish consumption by women of childbearing age over the 6-year period between 1999 and 2004. Our evaluation of NHANES fish intake data indicated no differences in the mean frequency or amount of particular fish and shellfish species consumed. However, the estimated 30-day Hg intake decreased at the 90th percentile and higher, whereas total fish consumption did not, which suggests a shift in fish species consumed. The BHg data indicated a reduction of the higher end of the distribution of BHg between the first 2-year interval (the 1999 and 2000 examinees) compared with the subsequent 4-year interval (the 2001–2004 examinees).The basis for these differences could possibly reflect spillover from the federal fish advisory program  in termsThe four fish species listed in the federal advisory  were raDuring the past decade, the U.S. EPA initiated substantial interventions aimed to reduce Hg releases and exposures  and issuSignificant geographic differences in BHg concentrations occurred within the United States: We found highest exposures in coastal areas and the Northeast census region. In the Northeast, 19% of women had BHg concentrations ≥ 3.5 μg/L. The highest 5% of BHg concentrations exceeded 8.2 μg/L in the Northeast and 7.2 μg/L in coastal areas, concentrations more than twice the 3.5 μg/L level of concern. BHg levels were predicted by the quantity and type of fish consumed. Over the 6-year period (1999–2004), the frequency of elevated BHg levels among women of childbearing age declined without a significant change in quantities of fish and shellfish consumed. This pattern suggests a more discerning series of choices in type of fish eaten rather than an overall reduction in fish consumption. Within all geographic regions, women at highest risk of elevated Hg exposures were more affluent and more likely to be of Asian or island ethnicity.


We report here the use of multiplex fluorescent polymerase chain reaction (PCR) for quantitative allele loss detection using microsatellites with 2-5 base pair repeat motifs. Allele loss of APC, DCC, p53 and RB1 in colorectal tumours has been reported previously using a variety of methods. However, not all workers used intragenic markers. We have used microsatellite polymorphisms which map within, or are closely linked to, these tumour-suppressor gene loci in order to determine whether these loci are indeed the targets for alteration in colorectal cancer. In addition, we have assayed two other tumour-suppressor genes, WT1 and NF1, to see whether they play a role in colorectal carcinogenesis. The putative metastasis-suppressor gene, NM23, was also investigated since there have been conflicting reports about its involvement in colorectal carcinogenesis. Allele loss was detected at the DCC (29%), p53 (66%), RB1 (50%) and NF1 (14%) loci and in the APC/MCC region (50%), but not at the WT1 or NM23 loci. These rapid, and mostly gene-specific, fluorescent multiplex PCR assays for allele loss detection could be modified to devise a single molecular diagnostic test for the important lesions in colorectal cancer.


To determine the relative sufficiency of paired aspirates of posterior uveal melanomas obtained by FNAB for cytopathology and GEP, and their prognostic significance for predicting death from metastasis.Prospective non-randomized IRB-approved single-center longitudinal clinical study of 159 patients with posterior uveal melanoma sampled by FNAB in at least two tumor sites between 09/2007 and 12/2010. Cases were analyzed with regard to sufficiency of the obtained aspirates for cytopathologic classification and GEP classification. Statistical strength of associations between variables and GEP class was computed using Chi-square test. Cumulative actuarial survival curves of subgroups of these patients based on their cytopathologic versus GEP-assigned categories were computed by the Kaplan–Meier method. The endpoint for this survival analysis was death from metastatic uveal melanoma.P < 0.001). Six of 34 tumors (17.6 %) that yielded an insufficient aspirate for cytopathologic diagnosis were categorized as GEP class 2, while 43 of 125 tumors (34.7 %) that yielded a sufficient aspirate for cytopathologic diagnosis were categorized as GEP class 2. To date, 14 of the 49 patients with a GEP class 2 tumor (28.6 %) but only five of the 109 patients with a GEP class 1 tumor (5.6 %) have developed metastasis. Fifteen of 125 patients (12 %) whose tumors yielded sufficient aspirates for cytopathologic classification but only four of 34 patients (11.8 %) whose tumors yielded insufficient aspirates for cytopathologic classification developed metastasis. The median post-biopsy follow-up time for surviving patients in this series was 32.5 months. Cumulative actuarial 5-year probability of death from metastasis 14.1 % for those with an insufficient aspirate for cytopathologic classification versus 22.4 % for those with a sufficient aspirate for cytopathologic classification (log rank P = 0.68). In contrast, the cumulative actuarial 5-year probability of metastatic death was 8.0 % for those with an insufficient/unsatisfactory aspirate for GEP classification or GEP class 1 tumor, versus 45.0 % for those with a GEP class 2 tumor (log rank P = 0.005).FNAB aspirates were insufficient for cytopathologic classification in 34 of 159 cases (21.9 %). In contrast, FNAB aspirates were insufficient for GEP classification in only one of 159 cases (0.6 %). This difference is statistically significant (This study confirmed that GEP classification of posterior uveal melanoma cells obtained by FNAB is feasible in almost all cases, including most in which FNAB yields an insufficient aspirate for cytodiagnosis. The study also confirmed that GEP classification is substantially better than cytologic classification for predicting subsequent metastasis and metastatic death. While ophthalmologists commonly regard primary uveal melanoma as a purely ophthalmic disorder, the reality is that this condition is a systemic disease that conveys to the affected person a substantial risk of metastasis and metastatic death. Although multiple clinical  and histAccording to published peer-reviewed data, primary uveal melanomas evaluated by GEP cluster into two distinct subgroups . GEP claThis study was conducted with the purpose of determining the relative sufficiency of paired aspirates of posterior uveal melanomas obtained by fine needle aspiration biopsy (FNAB) for cytopathologic classification and gene expression profile (GEP) classification, and to determine the relative prognostic significance of these different classifications for predicting subsequent patient death from metastasis.n = 134 cases, 81.3 %) or the Castle Biosciences, Inc., reference laboratory   for gene expression profiling and GEP classification. GEP testing on all cases in this series was performed using a previously described PCR-based 15-gene assay comprising 12 discriminating genes and three endogenous control genes [Our study was a prospective, non-randomized, IRB-approved, single-center longitudinal clinical study of 159 patients with primary posterior uveal melanoma sampled by FNAB between September 2007 and December 2010. All tumors in this series were sampled in at least two separate sites, and most were sampled in four separate sites using previously published FNAB techniques , 12. TheAll patient data was retrieved, and all cases were analyzed with regard to sufficiency of the obtained aspirates for cytopathologic classification and GEP classification. Aspirates were classified cytologicaly as insufficient for cytopathologic diagnosis, melanocytic nevus, borderline melanocytic tumor, spindle cell melanoma, mixed cell melanoma, unspecified uveal melanoma, epithelioid melanoma, or necrotic melanoma. Aspirates were categorized by GEP as class 1, class 2, or failed .Cumulative actuarial survival curves of subgroups of these patients based on their cytopathologic versus GEP-assigned categories were computed by the Kaplan–Meier method. The endpoint of this survival analysis was death from metastatic uveal melanoma. Significance of differences between the curves was evaluated using the log rank test. Statistical strength of associations between the studied clinical variables and GEP class of the tumor was evaluated using cross-table analysis with computation of the chi-squared statistic. An alpha level of 0.05 was selected as the cutoff value for assigning statistical significance of differences identified by this analysis. All statistical analyses were performed using Statistical Package for the Social Sciences (SPSS) version 11.1 for Windows.P = 0.137). The tumors studied had basal diameters that ranged from 4.5 to 22 mm , and thicknesses from 1.7 to 16 mm . Tumors were located anywhere from 0 to 23 mm from the disc  and from 0 to 21 mm from the fovea . The correlation between largest basal diameter and GEP classification of the melanocytic tumors studied is revealed in Table Our study group consisted of 159 patients whose ages ranged from 17 to 87 years, with a median age of 60.6 years and mean age of 60.9 years. There was no correlation between patient age and GEP classification of the tumor (P < 0.001).In cases of sufficient aspirate for cytological diagnosis, one was classified as a melanocytic nevus (0.6 %), ten were classified as a borderline melanocytic tumor (6.3 %), 37 were spindle cell melanoma (23.3 %), 34 were mixed cell melanoma (21.4 %), eight were unspecified melanomas (5.0 %), and 35 were epithelioid cell or necrotic melanoma (22 %). The tumor in the 158 successful cases was categorized as GEP class 1 in 109 (69.0 % %) and GEP class 2 in 49 (31.0 %) (Table FNAB yielded an insufficient aspirate for cytopathologic classification in 34 of the 159 cases (21.4 %). In contrast, the FNAB aspirates were insufficient (or unsatisfactory) for GEP classification in only one of the 159 cases (0.6 %). This difference is highly statistically significant (Six of the 34 cases (17.6 %) with an insufficient aspirate for cytopathologic classification proved to be Class 2 tumors by GEP. Four of these six patients have died of choroidal melanoma metastasis to date, and the other two have been seen in recent clinical follow-up. Both patients remain healthy and are metastasis-free. In contrast, 43 of the 125 cases (34.7 %) that yielded a sufficient FNAB aspirate for cytopathologic classification proved to be GEP Class 2 tumors (Table P = 0.68)   with a GEP class 2 tumor but only five of the 109 patients (1.8 %) with a GEP class 1 tumor have developed metastasis from their uveal melanoma Table . In cont2.4 % witRecognizing the substantial potential for primary uveal melanomas to metastasize and kill the affected host , ophthalmologists and medical oncologists have long desired a clinically useful method of testing patients with this tumor that (1) has a high probability of classification success in all patients tested, and (2) distinguishes reliably between those at high versus low risk of developing metastasis. Gene expression profiling of the cells of the primary intraocular tumor obtained by FNAB seems to fulfill most of the desires , 15.Because the clinical and cytologic prognostic factors for survival of uveal melanoma patients have been extensively discussed , 16, 17,Largest basal diameter of the studied tumors presented a significant correlation with GEP classification. Smaller tumors were much more likely to be GEP class 1; however, in the larger tumors the difference in distribution between class 1 and class 2 tumors by GEP was not as distinguishable. Once again, this result may be due to our sample being composed of smaller tumors, and perhaps a larger sample size would show a more significant trend in the distribution GEP class among the medium and larger tumors.Tumors involving the ciliary body have been known to have a worse survival prognosis . In our The yield of FNAB is an important topic to be considered. Many authors have pointed out the limited specimen yield of FNAB using smaller gauge needles (25- and 27-G) as a limitation for the widespread use of FNAB. Conversely, FNAB yield for cytology has been reported to be lower among smaller thinner tumors . AlthougFurthermore, our study has shown a definite trend in correlation between insufficiency of aspirates and class 1 tumors. An association between absence of epithelioid cells and GEP class 1 tumors cannot be established at this point. Again, definitive correlations may not be present due to our sample size.This study confirmed that GEP classification of frozen posterior uveal melanoma cells obtained by FNAB is feasible in almost all cases, including most in which FNAB yields an insufficient aspirate for cytopathologic diagnosis. The study also confirmed that GEP classification is substantially better than cytopathologic classification for predicting subsequent metastasis and metastatic death.


Standard precautions (SP) aim to protect healthcare workers (HCW) and patients from infectious diseases arising from bloodborne pathogens and reduce the risk of cross transmission of micro-organisms. They must be applied in all circumstances, regardless of the infectious status of the patient.The objectives were to assess: 1) institutional policies for SP promotion; 2) available resources for SP implementation; and 3) education of HCW and their compliance with SP.st and December 31st 2011, supported by the Ministry of Health. Inclusion criteria were voluntary public and private hospitals in France, medical, surgical and medico-technical wards therein and HCW working with patients in these wards. Self-assessment questionnaires were administered at three levels: institutional, ward and HCW. At institutional and ward levels, results were given as a percentage of objectives attained; at professional level, percentages of responses reported as “never”, “sometimes”, “often” or “always” were calculated for each question.The study was a mixed audit of procedures, resources and attitudes. It was conducted between February 1A total of 1,599 hospitals participated, including 14,968 wards and 203,840 HCW. At institutional level, the overall score was 88%, covering: SP promotion (91%), procedures (99%) and SP evaluation (63%). At ward level, the overall score was 94%, covering: procedures (95%) and resources (93%). Among the 165,722 (81.3%) HCW who reported having participated in a training session on SP, 69.6% had had it in the last five years. A total of 88.1% of HCW knew where to find the appropriate written procedure in the event of a blood exposure. HCW reported the best compliance for glove changing between two patients . The less respected criteria were glove use for intramuscular or subcutaneous injection and eye protection use in the event of blood exposure risk .No study on SP exists in literature which includes such a large participation as this one. It will form a refence basis leading to actions for improvement at local and national level.None declared.


Varicose veins are elongated and dilated saphenous veins. Despite the high prevalence of this disease, its pathogenesis remains unclear.2 during the vascular wall remodeling of human varicose veins.In this study, we investigated the control of matrix metalloproteinases (MMPs) expression by prostaglandin (PG)E2 metabolism were analyzed in all veins. cPGES protein was absent while its mRNA was weakly expressed. mPGES-2 expression was similar in the different saphenous veins. mPGES-1 mRNA and protein were detected in healthy veins and a significant decrease was found in LDv. Additionally, 15-hydroxyprostaglandin dehydrogenase (15-PGDH), responsible for PGE2 degradation, was over-expressed in varicose veins. These variations in mPGES-1 and 15-PGDH density account for the decreased PGE2 level observed in varicose veins. Furthermore, a significant decrease in PGE2 receptor (EP4) levels was also found in SDv and LDv. Active MMP-1 and total MMP-2 concentrations were significantly decreased in varicose veins while the tissue inhibitors of metalloproteinases (TIMP -1 and -2), were significantly increased, probably explaining the increased collagen content found in LDv. Finally, the MMP/TIMP ratio is restored by exogenous PGE2 in varicose veins and reduced in presence of an EP4 receptor antagonist in healthy veins.Varicose  and large diameter (LDv)) and healthy saphenous veins (SV) were obtained after surgery. Microsomal and cytosolic PGE-synthases (mPGES and cPGES) protein and mRNA responsible for PGE2 could be responsible for the vascular wall thickening in human varicose veins. This mechanism could be protective, strengthening the vascular wall in order to counteract venous stasis.In conclusion, PGE Varicose saphenous veins are characterized by venous backflow and blood stagnation. 2via selective activation of EP1-4 receptor subtypes is involved in the control of vascular tone, 2 is synthesized from arachidonic acid (AA) through the enzymatic activities of two cyclooxygenases (COX-1 and/or COX-2) and three PGE synthases (PGES). 2 is degraded by 15-hydroxyprostaglandin dehydrogenase (15-PGDH), the only enzyme responsible for its catabolism. Among the three PGES that specifically catalyze the final step of PGE2 biosynthesis; two are constitutive: microsomal (mPGES-2) and cytosolic (cPGES). 2 production. mPGES-1 and COX-2 expression are generally co-induced by inflammatory cytokines such as IL-1β. The metabolism and the effects of bioactive lipids like prostanoids (prostaglandins (PG) and thromboxane) has been rarely investigated in the context of varicose veins. Prostanoids are produced by most blood and vascular cell types. 2 modulates vascular wall remodeling mediated by the matrix metalloproteinases (MMPs). The renewal of extracellular matrix (ECM) by MMP 2 on the expression/activation of MMPs. 2 activates several MMPs via EP2/EP4-receptor stimulation in human endometriotic epithelial and stromal cells. As observed during aneurysm formation or in the pathogenesis of endometriosis, 2 in the mechanism involved in the formation of varicose veins. We have investigated how PGE2 and the enzymes responsible for its metabolism could participate in venous wall remodeling via MMP, TIMP and collagen deposition. Specifically, this study was designed to analyse the pathology of varicose veins. For each patient with varicose veins (C2 stage) removed by surgery, we compared a dilated segment and a non-dilated segment. These two samples were compared with healthy saphenous veins (SV). An increase in diameter between segments of the healthy SV and the lesser dilated varicose vein can be observed. Therefore, our hypothesis is that the non-dilated varicose vein segment represents an intermediate stage of the disease. In this way, our study addresses the evolution of varicose pathology.The aim of this study was to investigate the role of PGEFifteen healthy SV from calf patients undergoing bypass surgery  were obtained. In most European countries, varicose veins are still frequently removed by conventional surgery. Varicose veins  from patients undergoing vein stripping  were obtained at Bichat Hospital  with patients’ verbal and written informed consent. The consent form, explaining all the procedure, was approved by the Bichat ethic committee. The investigation conforms to the principles outlined in the declaration of Helsinki as these tissues were anonymized. All research programs involving the use of human tissue were approved and supported by the National Institute for Health and Medical Research (INSERM) ethics committee and these tissues are considered as surgical waste in accordance with French ethical laws (L.1211-3–L.1211-9). Following harvesting, veins were rapidly placed in ice-cold saline. After surgical use, non-used healthy SV (∼3 mm) or varicose vein segments were immediately transported to the laboratory. All veins were processed within two hours after excision. They were cleaned of adipose tissue and blood. Varicose veins were separated into two different segments, of small diameter  and of large diameter . Saphenous veins obtained after bypass surgery are considered as healthy since patients were not under NSAIDs medication and veins were checked using sonographic ultrasound studies. All preparations were used with intact adventitial layers.2, MMPs or TIMPs, fresh vein samples were incubated at 37°C either for 24 h (70 mg wet weight tissue/mL solution) in RPMI solution  with 5% CO2 or for 30 min in Tyrode’s solution.with 5% CO2 in O2. RPMI solution always contained antibiotics  and antimycotic . The fresh venous preparations were incubated for 24 h in RMPI+antibiotics solution with or without pharmacological treatments (EP receptor agonist or antagonist). The fresh venous preparations were incubated for 30 min in Tyrode’solution with or without (arachidonic acid +/− glutathione). Supernatants were harvested and frozen until use.For Western blotting, fresh vein samples were ground in liquid nitrogen and homogenized in a RIPA solution as previously described. Protein analysis was evaluated as previously described. 2 in the supernatant of healthy SV or varicose veins was determined using an enzyme immunoassay (EIA) kit (Cayman). Frozen supernatants were used and diluted with the assay buffer. The PGE2 concentration was determined according to the instructions provided with the kit. In addition, 30 min incubations in presence of arachidonic acid  and with or without glutathione  The concentration of PGE2  or an EP4 receptor antagonist GW627368X MMP and TIMP protein levels were measured in the supernatant of vascular preparations using commercial kits . Tissues were incubated in absence or presence of PGEMasson’s trichrome staining was used for detection of several collagen and general morphology Due to the specificity of the experiments, several normalization procedures were performed (Supplementary data). For specific protein content measured by Western blot, the level of protein expression was normalised by the α-actin content. Optical density (OD) for the different bands was measured by Scion Image®. For EIA measurements, normalisation was done by correcting for tissue wet weight. Collagen content were measured by calculating the optical density (pixel number) with Photoshop® and normalized by the total area of the vein sections.Statistical analyses were performed using the program SigmaStat® . All data are presented as means ± sem derived from (n) patients. A one-way-ANOVA test (followed by the Tukey post-hoc test) or a paired t-test was used and a P-value <0.05 was considered as statistically significant. Linear regression and the Pearson test were used for correlation analysis (SigmaStat®).Expression of PGES isomers was measured using Western blot analysis and normalized to α-actin ; for mPGUsing Western blot analysis, a 29 kDa band corresponding to 15-PGDH was observed in the protein extracts of saphenous veins corresponding to the standard band . A significant increase (∼188%) was found in varicose veins  as compared to healthy SV n = 5; .2 concentrations in the solution incubated for 24 h , in the presence or absence of glutathione (2.5 mmol/L) . This decreased content in PGE2 in varicose veins was associated with an increased production of TxA2 and PGD2, measured as their stable metabolites TxB2 and 15d-PGJ2, respectively  was observed for the EP4 receptor  in the h2 led to the activation of MMP-1 in varicose veins (SDv and LDv) to the same level as in healthy SV. The saphenous veins incubated with an EP4 antagonist showed a significant decrease in MMP-1 activation (2 induced a decreased TIMP expression in varicose veins (SDv and LDv) whereas the healthy veins incubated with the EP4 antagonist showed a significant increase in TIMP. The EIA measurement for TIMP-2  and varicose (n = 5) SV . A signi2 plays a major role in vascular wall remodeling and in collagen over-expression. The reduced synthesis (via decreased mPGES-1) and/or increased degradation (via increased 15-PGDH) of PGE2 are responsible for its lower concentration in human varicose saphenous veins when compared to healthy ones. This reduced PGE2 content and the lower density of its receptor (EP4) are responsible for the down-regulation of the MMP/TIMP ratio in varicose veins. The consequence of this biological cascade is a reduction of active collagenase content and an accumulation of collagen in the vascular wall of varicose veins and could explain the intima hyperplasia and the thickening observed in the varicose wall Our results demonstrate that in the varicose veins, PGEvia NF-κB. 2 are shown. We also showed a significant decrease (50–75%) in mPGES-1, at both protein and mRNA levels, in LDv compared to the other segments (SV and SDv). This decreased content could be due to several mechanisms such as an increased of PGD2 metabolite, 15d-PGJ2 (as observed in 2 are in favour of a reduced concentration of this prostaglandin in varicose veins. This effect is not due to a decreased expression of COX-1 since the protein content was increased (Supplementary data). This dysregulation of PGE2 production led to a reorganisation of PG synthesis such as an increased thromboxane A2 and PGD2 production  as compared with SV. For example, the EP4-mediated vasodilation previously reported in human healthy saphenous veins 2 and EP4 receptor expression.In our human saphenous vein preparations, the accumulation of released PGEMMP -1, -2, -9, some enzymes responsible for ECM degradation, and/or their inhibitors TIMP-1, -2, have been measured in human varicose veins and the results of many studies are controversial . (A) Histogram represent western blot analysis for internal mammary arteries  for cPGES. (B) Representative samples. Optical density  was measured by Scion Image® and the mean normalized by actin.(TIF)Click here for additional data file.Figure S2Dysregulation in PGES mRNA. mRNA expression in human small and large diameter varicosities  and healthy saphenous veins . mRNA levels of prostaglandin E synthases  were determined by Real-Time PCR and normalized by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA level; * P<0.05 as determined by one-way-ANOVA followed by the Tukey post-hoc test and by a paired t-test for varicose veins.(TIF)Click here for additional data file.Figure S3Increased COX-1 protein in varicose veins. Protein measurements (A), representative samples of Western blot of cyclooxygenases (COX-1 or COX-2) in human small and large diameter varicosities  and healthy saphenous veins . Standards (Std) are Western ready controls from Cayman. Histogram (B) represents Western blot quantification of COX-1 corresponding band. Optical density  was measured by Scion Image® and the mean normalized by actin; * P<0.05 as determined by one-way-ANOVA followed by the Tukey post-hoc test and by a paired t-test for varicose veins.(TIF)Click here for additional data file.Figure S4Decreased PGE2 content in 30 min. PGE2 content in human small and large diameter varicosities  and healthy saphenous veins . Values were determined by EIA in supernatants after 30 min of incubation of the venous preparations in Tyrode solution. Results are normalized by tissue wet weight; * P<0.05 as determined by one-way-ANOVA followed by the Tukey post-hoc test and by a paired t-test for varicose veins.(TIF)Click here for additional data file.Figure S5Prostanoids expression. Thromboxane (Tx) B2 and 15d-PGJ2 content in human small and large diameter varicosities  and healthy saphenous veins . Values were determined by EIA in supernatants after 24 hours of incubation of the venous preparations in RPMI solution. Results are normalized by tissue wet weight; * P<0.05 as determined by one-way-ANOVA followed by the Tukey post-hoc test and by a paired t-test for varicose veins.(TIF)Click here for additional data file.File S1Supporting information about experimentals protocols. Table S1. Primers used for Real-Time PCR, s: sense, as: anti sense.(DOC)Click here for additional data file.


Identification of genes responsible for medically important traits is a major challenge in human genetics. Due to the genetic heterogeneity of hearing loss, targeted DNA capture and massively parallel sequencing are ideal tools to address this challenge. Our subjects for genome analysis are Israeli Jewish and Palestinian Arab families with hearing loss that varies in mode of inheritance and severity.CDH23, MYO15A, TECTA, TMC1, and WFS1. Critical mutations of the probands co-segregated with hearing loss. Screening of additional families in a relevant population was performed. TMC1 p.S647P proved to be a founder allele, contributing to 34% of genetic hearing loss in the Moroccan Jewish population.A custom 1.46 MB design of cRNA oligonucleotides was constructed containing 246 genes responsible for either human or mouse deafness. Paired-end libraries were prepared from 11 probands and bar-coded multiplexed samples were sequenced to high depth of coverage. Rare single base pair and indel variants were identified by filtering sequence reads against polymorphisms in dbSNP132 and the 1000 Genomes Project. We identified deleterious mutations in Critical mutations were identified in 6 of the 11 original probands and their families, leading to the identification of causative alleles in 20 additional probands and their families. The integration of genomic analysis into early clinical diagnosis of hearing loss will enable prediction of related phenotypes and enhance rehabilitation. Characterization of the proteins encoded by these genes will enable an understanding of the biological mechanisms involved in hearing loss. Clinical diagnosis is the cornerstone for treatment of human disease. Elucidation of the genetic basis of human disease provides crucial information for diagnostics, and for understanding mechanisms of disease progression and options for treatment. Hence, determination of mutations responsible for genetically heterogeneous diseases has been a major goal in genomic medicine. Deafness is such a condition, with 61 nuclear genes identified thus far for non-syndromic sensorineural hearing impairment [GJB2, which is responsible for 27% of congenital hearing loss among Israeli Jews [Most early-onset hearing loss is genetic . Of geneeli Jews  and 14% eli Jews . Each ofSTRC [OTOA [We developed a targeted capture pool for identifying mutations in all known human genes and human orthologues of mouse genes responsible for syndromic or non-syndromic hearing loss. Targets were 82 human protein-coding genes, two human microRNAs and the human orthologues of 162 genes associated with deafness in the mouse  were analyzed with paired-end sequencing at a read length of 2 × 72 bp, using the Illumina Genome Analyzer IIx. Across the 1.43 MB of captured targets, median base coverage was 757 × to 2,080 ×, with 95% and 92% of targeted bases covered by more than 10 or 30 reads, respectively. We aligned reads to the human reference genome sequence (hg19) and generated SNP and indel calls for all samples. Rare variants were identified by filtering against dbSNP132, the 1000 Genomes project and additional filters  and classified by predicted effect on the protein, as described in Materials and methods.In each of the 11 probands, multiple potentially functional variants of predicted damaging effect were identified by our approach and validated by Sanger sequencing /104, or 0.34 , was supported by 50% (43/86) of reads and is predicted to be damaging by PolyPhen2 and SIFT. The other MYO15A variant was cryptic. It was read as two single base-pair substitutions 2 bp apart, at chr17:18,022,486 C > G and chr17:18,022,488 G > C, but each variant was supported by only 25% of reads. In our experience, two apparently adjacent or nearly adjacent single base-pair variants with similar numbers of reads, each with weak support, may reflect an underlying insertion or deletion. We sequenced MYO15A exon 2 containing these variant sites and detected a 2-bp deletion MYO15A c.373delCG (p.R125VfsX101). MYO15A p.R2728H and MYO15A c.373delCG co-segregated with hearing loss in the family. MYO15A, which encodes a myosin expressed in the cochlea, harbors many mutations worldwide responsible for hearing loss [MYO15A c.373delCG has been described previously.Family Z421 is of Jewish Ashkenazi origin. Hearing loss in the family is consistent with recessive inheritance , a novel mutation predicted to be damaging by Polyphen2 and SIFT   [Family K13576 is of Ashkenazi Jewish origin. Hearing loss in the family is dominant Figure . Audiogr01.0020) ,18. Hete01.0020)  with a sTECTA c.5597C > T (p.T1866M)  Tables  and 3, wIn addition to the probands described above, in five other probands of Palestinian Arab origin , multiple variants were identified by capture and sequencing, and validated by Sanger sequencing, but none co-segregated with hearing loss in the families Table . In thesThe goal of our study was to apply DNA capture and MPS to identify inherited mutations involved in hearing loss. We designed oligonucleotides to capture the exons and regulatory regions of 246 genes involved in hearing loss, in human or in mouse. The inclusion of genes thus far known to be involved in deafness in the mouse is based on the observation that many genes for human deafness are responsible for mouse deafness as well ,24. AmonComprehensive targeted enrichment and MPS has been employed previously for non-syndromic hearing loss . Our appGJB2 mutations, and on occasion, DNA sequencing of the GJB2 coding region. In some clinics, screening for the relevant mutations in other genes on the basis of ethnic origin, audiological tests, family history, personal history and findings from physical examination may be performed. Comprehensive testing for genes with mutations common in other populations, such as TMC1 [MYO15A [SLC26A4 [To date, routine clinical diagnostic tests for deafness in the Middle East have consisted of restriction enzyme analysis of the two common  as TMC1 ,26, MYO1[SLC26A4 , is not A major challenge for mutation discovery is determining which variants are potentially causative and which are likely benign. This is particularly difficult when sequencing populations that are not well represented in dbSNP. A novel variant may represent a previously undiscovered common population-specific polymorphism or a truly private mutation. Sequencing even a small number of samples (say 100) from the same ethnic background serves as a very effective filter. In our study, many variants not in dbSNP were nonetheless common in our populations and could be ruled out as causative mutations  of hearing impaired Jews of Moroccan origin. A single DNA sample of a Moroccan Jewish proband, evaluated by this approach, led to the discovery of four mutations, two of them novel, and solved the cause of hearing loss of an additional 20 families. The TMC1 gene is the sixth most common cause of recessive hearing loss worldwide [TMC1 gene [TMC1 is one of the genes most frequently involved in deafness. Based on these results, we recommend that all Israeli Jewish probands of Moroccan ancestry be screened for the four TMC1 mutations, as well as for the most common GJB2 mutations, prior to conducting MPS. An immediate result of these findings is that screening for TMC1 mutations will become routine in Israel for all hearing impaired patients of Moroccan Jewish ancestry.For the Israeli deaf population of Moroccan Jewish ancestry, this study has substantial clinical implications, as the orldwide . The twoMC1 gene . In someMC1 gene  and TurkMC1 gene , as IsraCDH23, MYO15A, WFS1, and TECTA - that are known to be responsible for hearing loss but are not routinely evaluated, largely because of their size. Targeted MPS makes it feasible to screen large genes that have heretofore been largely untested. As sequencing chemistry improves, we believe it will be feasible to multiplex 12 samples per lane and still maintain a high coverage (> 200 ×). It will thus become even more straightforward to screen comprehensively for all known hearing loss genes.Novel mutations were identified in multiple other genes - Of the six Palestinian families enrolled in this study, a causative mutation was found in only one. This result is probably due to two factors. First, familial hearing loss in the Palestinian population has been very thoroughly investigated for more than a decade, with the discovery of many critical genes and the characterization of the mutational spectra of these genes as they were identified Multiple mutations responsible for hearing loss were identified by the combination of targeted capture and MPS technology. Screening multiple families for alleles first identified in one proband led to the identification of causative alleles for deafness in a total of 25 of 163 families. The approach described here exploits the high throughput of targeted MPS to make a single fully comprehensive test for all known deafness genes. Although we applied it within the context of familial hearing loss, the test could also be used in cases of isolated deafness. This strategy for clinical and genetic diagnosis will enable prediction of phenotypes and enhance rehabilitation. Characterization of the proteins encoded by these genes will enable a comprehensive understanding of the biological mechanisms involved in the pathophysiology of hearing loss.The study was approved by the Helsinki Committees of Tel Aviv University, the Israel Ministry of Health, the Human Subjects Committees of Bethlehem University, and the Committee for Protection of Human Subjects of the University of Washington (protocol 33486). Eleven probands and both affected and unaffected relatives in their families were ascertained. A medical history was collected, including degree of hearing loss, age at onset, evolution of hearing impairment, symmetry of the hearing impairment, use of hearing aids, presence of tinnitus, medication, noise exposure, pathologic changes in the ear, other relevant clinical manifestations, family history and consanguinity. The only inclusion criteria for our study were hearing loss and family history. Blood was drawn when subjects signed committee-approved consent forms for DNA extraction, and genomic DNA was extracted.GJB2 [GJB6 [PCDH15 [USH1C [MYO3A [SLC26A4 [POU4F3 [TJP2 [LOXHD1 [CDH23, MYO7A, MYO15A, OTOF, PJVK, SLC26A4, TECTA, TMHS, TMPRSS3, OTOA, PTPRQ, and GPSM2 [All subjects were tested for GJB2  by standB2 [GJB6 , PCDH15  [PCDH15 , USH1C [5 [USH1C , MYO3A [C [MYO3A , SLC26A4[SLC26A4 , POU4F3  [POU4F3 , the invF3 [TJP2 , and LOX [LOXHD1 . All knoExons and the flanking 40 bp into introns of 246 human genes were selected for capture and sequencing. The 246 genes are listed in Additional file Paired-end libraries were prepared by shearing 3 μg of germline DNA to a peak size of 200 bp using a Covaris S2. DNA was cleaned with AmpPure XP beads , end repaired, A-tailed and ligated to Illumina indexing-specific paired-end adapters. The libraries were amplified for five cycles with flanking primers (forward primer PE 1.0 and reverse primer SureSelect Indexing Pre-Capture PCR). The purified amplified library (500 ng) was then hybridized to the custom biotinylated cRNA oligonucleotides for 24 hours at 65°C. The biotinylated cRNA-DNA hybrids were purified with streptavidin-conjugated magnetic beads, washed, and the cRNA probes were digested, following cleaning of the captured DNA fragments with AmpPure XP beads. Barcode sequences for multiplex sequencing were added to the captured DNA samples, and a post capture PCR was performed for 14 cycles. The libraries were prepared using reagents from Illumina (Genomic DNA Sample Preparation Kit and Multiplexing Sample Preparation Oligonucleotide Kit) and Agilent (SureSelect Target Enrichment System Kit), according to Agilent's instructions. The final concentration of each captured library was determined by a Qubit fluorometer and multiple aliquots diluted to 0.5 ng/μl were analyzed on a high sensitivity chip with a Bioanalyzer 2100.A final DNA concentration of 12 pM was used to carry out cluster amplification on v4 Illumina flow cells with an Illumina cluster generator instrument. We used a 2 × 72-bp paired-end recipe plus a third read to sequence the 6-bp index to sequence 11 captured library samples in total   and MAQ The Placental Mammal Basewise Conservation by PhyloP  score for the consensus nucleotide in each SNP was obtained from the UCSC Genome Browser, and variants with a score < 0.9 were considered as non-conserved and discarded from the SNP lists. Since we sequenced DNA samples of 11 probands from similar ethnic groups, we also counted the number of probands that carry each variant, finding many novel variants that are common in the Jewish and/or Palestinian ethnic groups, although not included in dbSNP132, which are most probably non-damaging variants. For variants of conserved nucleotides that present in up to three probands, we also checked if this variant was already reported in the 1000 Genomes project or in other published genomes from hearing humans.The effect of rare or private non-synonymous SNPs was assessed by the PolyPhen-2  HumVar score  and SIFTSequencing was performed using the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit  and an ABI 377 DNA sequencer.CDH23 c.7903G > T (p.V2635F); TMC1 c.1810C > T (p.R604X), c.1939T > C (p.S647P) and c.1210T > C, W404R; MYO15A c.8183G > A (p.R2728H) and c.373delCG (p.R125VfsX101); and TECTA c.5597C > T (p.T1866M)  and WFS1 c.2765G > A (p.E864K)  Table of indels appearing in four or more probands in our population (n = 11). (b) Table of SNPs in four or more probands in our population (n = 11).Click here for fileTable of primers and restriction enzyme digestion assays.Click here for file


We tested whether a diet supplemented with CoQ10 ameliorates oxidative stress and mitochondrial alteration, as well as promotes retinal ganglion cell (RGC) survival in ischemic retina induced by intraocular pressure elevation. A CoQ10 significantly promoted RGC survival at 2 weeks after ischemia. Superoxide dismutase 2 (SOD2) and heme oxygenase-1 (HO-1) expression were significantly increased at 12 h after ischemic injury. In contrast, the CoQ10 significantly prevented the upregulation of SOD2 and HO-1 protein expression in ischemic retina. In addition, the CoQ10 significantly blocked activation of astroglial and microglial cells in ischemic retina. Interestingly, the CoQ10 blocked apoptosis by decreasing caspase-3 protein expression in ischemic retina. Bax and phosphorylated Bad (pBad) protein expression were significantly increased in ischemic retina at 12 h. Interestingly, while CoQ10 significantly decreased Bax protein expression in ischemic retina, CoQ10 showed greater increase of pBad protein expression. Of interest, ischemic injury significantly increased mitochondrial transcription factor A (Tfam) protein expression in the retina at 12 h, however, CoQ10 significantly preserved Tfam protein expression in ischemic retina. Interestingly, there were no differences in mitochondrial DNA content among control- or CoQ10-treated groups. Our findings demonstrate that CoQ10 protects RGCs against oxidative stress by modulating the Bax/Bad-mediated mitochondrial apoptotic pathway as well as prevents mitochondrial alteration by preserving Tfam protein expression in ischemic retina. Our results suggest that CoQ10 may provide neuroprotection against oxidative stress-mediated mitochondrial alterations in ischemic retinal injury.Coenzyme Q10 (CoQThe online version of this article (doi:10.1007/s10495-013-0956-x) contains supplementary material, which is available to authorized users. Elevated intraocular pressure (IOP) is an important risk factor for retinal ganglion cell (RGC) death and optic nerve degeneration in retinal ischemia and glaucoma . GrowingOxidative stress has been implicated as an important pathophysiological mechanism for mitochondrial dysfunction-mediated retinal neurodegeneration including ischemic injury –7. SuperTfam have impaired mtDNA transcription and loss of mtDNA leads to bioenergetics dysfunction and embryonic lethality  [10 diet (n = 30 mice).CoQ20 mice]  and a grSix to 24 h after acute IOP elevation, light adapted mice were anesthetized with IP injection of mixture of ketamine/xylazine, as described, and then mice were perfused transcardially with 0.9 % saline followed by 4 % paraformaldehyde in 1X phosphate buffer saline . Both eyes enucleated and fixed in 4 % paraformaldehyde in PBS for 4 h at 4 °C. After several washes in PBS, the retinas were dissected and then dehydrated through graded ethanol solutions and embedded in polyester wax, as described previously .Retinas from enucleated eyes were dissected as flattened whole-mounts at 12 h or 2 weeks after ischemia–reperfusion. Retinas were immersed in PBS containing 30 % sucrose for 24 h at 4 °C. The retinas were blocked in PBS containing 3 % donkey serum, 1 % bovine serum albumin, 1 % fish gel and 0.1 % Triton X-100 for 1 h, and incubated with goat polyclonal anti-Brn3a antibody , a specific maker for RGCs , 29, and2, and then the number of RGCs were normalized per mm2. RGC densities were measured in 24 distinct areas  per condition by two investigators in a masked fashion, and the scores were averaged (n = 7 retinas/group). To further examine RGC survival between control and CoQ10 diet-treated non-ischemic retinas, RGC densities were automatically measured using ImageJ cell counting analysis .To count RGCs labeled with Brn3a, each retinal quadrant was divided into three zones by central, middle, and peripheral retina . Images were taken at 20×, covering an area of 0.344 mmImmunohistochemical staining of retinal cross sections at 12 h after transient ischemia was performed as previously described . Five sen = 3 retinas/group) was separated by PAGE and electrotransferred to polyvinylidenedifluoride membrane. The membrane was blocked with 5 % nonfat dry milk and 0.1 % Tween-20 in PBS for 1 h, incubated with rabbit polyclonal anti-SOD2 antibody , rabbit polyclonal anti-HO-1 antibody , monoclonal mouse anti-GFAP , rabbit polyclonal anti-Iba-1 antibody , mouse monoclonal anti-caspase antibody , rabbit polyclonal anti-Bax antibody , mouse monoclonal anti-phosphorylated Bad , goat polyclonal anti-Tfam antibody , rabbit polyclonal anti-Porin  and mouse monoclonal anti-actin antibody  for overnight at 4 °C. After several washes in Tween/PBS, the membranes were incubated with peroxidase-conjugated donkey anti-goat IgG , goat anti-rabbit IgG  or goat anti-mouse IgG , and developed using chemiluminescence detection . The scanned film Images were analysed by ImageJ  and band densities were normalized to the band densities for actin or porin.Following 6, 12 or 24 h after transient ischemia, the collected retinas were homogenized in a glass-Teflon Potter homogenizer in RIPA lysis buffer  containing complete protease inhibitors . Each sample  by two investigators in a masked fashion, and the scores were averaged.The retinal cross sections at 12 h after transient ischemia were incubated with proteinase K  for 10 min at 37 °C. After rinsing in PBS, the sections were incubated with terminal deoxynucleotidyl transferase plus nucleotide mixture in reaction buffer for 60 min at 37 °C  as previously described . Output data were obtained as Ct values and the difference of mtDNA content among samples was calculated using the comparative Ct method [MtDNA content of each sample at 12 h after transient ischemia was determined as described previously . Brieflyt method . ActB get test or one-way analysis of variance and the Bonferroni t test. P < 0.05 was considered to be statistically significant.Data were presented as the mean ± SD. Comparison of two or three experimental conditions was evaluated using the unpaired, two-tailed Student’s 10 (1 %)-supplemented diet treatment daily for 1 week before the induction of transient retinal ischemia and then continued diet treatment for 2 weeks  and 70.3 ± 5.0 mmHg in mice treated with CoQ10 diet (n = 30 mice) for 50 min  and heme oxygenase-1 (HO-1) protein expression using antibodies raised against SOD2 and HO-1. We observed that Increase of SOD2 protein expression was maximal 12 h later by 1.36 ± 0.04-fold in ischemic retina  , a marker for astroglial cells, or Iba1, a marker for microglial cells. We found that GFAP and Iba1 immunoreactivity were increased in activated astroglia and microglial cells in the GCL and nerve fiber layer of control diet-treated ischemic retina at 12 h, respectively .Since CoQ insults , 36, we 10 prevents apoptotic cell death in ischemic retina, we performed TUNEL staining. There were no TUNEL-positive cells in the retina of control mice treated control diet  [10 treatment could reduce the O2− production [SOD2 gene inhibits retinal damage by reducing oxidative stress and nitrosative stress [10 could be an important antioxidant for ameliorating oxidative stress-mediated neurodegeneration in ischemic retina. Studying the benefits of CoQ10-mediated blockade of oxidative stress or enhancement of antioxidant enzymes may be a novel strategy for protecting retinal neurons against ischemic injury.We previously reported that acute IOP elevation triggers the upregulation of SOD2 protein expression and this increase could be restored by brimonidine (BMD)-mediated blockade of glutamate excitotoxicity-induced oxidative stress in ischemic rat retina . Consistchondria , 45. It ia [2−)  and CoQ1oduction . Growinge stress , as welle stress . Moreovee stress , 51. Our10 treatment significantly promoted RGC survival as well as blocked activation of astroglial and microglial cells in ischemic retina. Astroglial or microglial activation coincides with RGC degeneration in the hypertensive retina of human, rat or mouse [10-mediated blockade of oxidative stress may indirectly reduce astroglial or microglial activation in the retina against elevated IOP-induced ischemic damage as a result of increased RGC survival. Regardless, it cannot be excluded that CoQ10 may prevent glial activation by blocking oxidative stress in astroglial or microglial cells in ischemic retina. CoQ10 is also neuroprotective in retinal glial cells against oxidative stress, since oxidative stress has remarkably linked to glial cell activation or reaction in the retina or optic nerve still needs to be ascertained [In the current study, we observed that CoQor mouse , 37–39. ertained , 52–56. ertained , 58. Alt10 promoted RGC survival in ischemic retina, we found that CoQ10 significantly decreased Bax, but increased pBad protein expression in ischemic retina. Bax is a pro-apoptotic member of the Bcl-2 family that is essential in many apoptotic pathways [10 promotes RGC survival against the mitochondria-related apoptotic pathway in ischemic retinal injury by decreasing Bax, but by increasing pBad protein expression. These results reflect the possibility that increased pBad expression may represent an endogenous repair mechanism against apoptotic pathway and that CoQ10 may contribute to the blockade of Bax-mediated increase of MPT or the promotion of mitochondrial homeostasis in ischemic injury. The MPTP opening leads to loss of mitochondrial membrane potential, mitochondrial swelling, rupture of the outer mitochondrial membrane and release of cytochrome c. Importantly, MPTP opening by calcium overload and oxidative stress leads to necrosis rather than apoptosis [10 may promote RGC survival by inhibiting not only apoptosis but also necrosis in ischemic retinal injury.Consistent with our result that CoQpathways , 60 as wpathways –63. Bax pathways , 65. Ourpoptosis , 67. Recpoptosis , 25. Tak10 decreases Tfam protein expression in ischemic retina at 12 h. Emerging evidence suggests that the Tfam and OXPHOS complex IV are rapidly increased in the early neurodegenerative events of neonatal hypoxic-ischemic brain injury, suggesting that Tfam may contribute to endogenous repair mechanism of injured brain neurons [Tfam there is impaired mtDNA transcription and mtDNA loss is triggered; this leads to mitochondrial bioenergetic dysfunction-mediated embryonic lethality [Ischemic injury triggers mitochondrial dysfunction including ROS induction as well as mtDNA damage, Tfam alteration or OXPHOS impairment in the central nervous system , 19, 68. neurons , 17. In 10-mediated preservation of Tfam expression may provide a potential mechanism for protecting RGCs against oxidative stress-induced mitochondrial dysfunction in ischemic retina. Future studies will examine the precise molecular mechanism underlying CoQ10-mediated Tfam preservation in ischemic retina. Although mtDNA is particularly susceptible to oxidative stress [10 diet-treated groups. Because it has been reported that Tfam regulates mtDNA copy numbers in mammals and the levels of Tfam correlate with the levels of mtDNA [Based on these results, we suggest that CoQe stress , it is uof mtDNA , 70, our10 promotes RGC survival in ischemic mouse retina by inhibiting oxidative stress and by blocking the Bax/Bad-mediated mitochondrial apoptotic pathway. Moreover, CoQ10 also preserves Tfam protein expression in ischemic mouse retina. Based on these observations, we suggest that CoQ10 may provide a promising therapeutic potential for ameliorating oxidative stress-mediated mitochondrial dysfunction in ischemic retinal injury.In conclusion, these results provide direct evidence that CoQSupplementary material 1 (DOCX 552 kb)Supplementary material 2 (DOCX 68 kb)Supplementary material 3 (DOCX 56 kb)Below is the link to the electronic supplementary material.