Many women of childbearing age are occupationally active, which leads to a large number of pregnancies potentially exposed to occupational exposures. Occupational noise has been identified as a risk factor for hearing impairment in adults. However, very few studies have assessed the effect of occupational noise on the fetus.The aim of this study was to investigate whether occupational exposure to noise during pregnancy is associated with hearing dysfunction in children.This population based cohort study included 1,422,333 single births in Sweden 1986–2008. Data on mothers’ occupation, smoking habits, age, ethnicity, body mass index, leave of absence, and socioeconomic factors were obtained from interviews performed by prenatal care unit staff at approximately 10 weeks of gestation and from national registers. Occupational noise exposure was classified by a job–exposure-matrix as < 75, 75–84, or ≥ 85 dBLAeq,8h. Diagnosed cases of hearing dysfunction  were identified from a register of specialized medical care. Cox proportional hazards models were used to estimate associations.In the full sample, containing a mixture of part-time and full-time workers during pregnancy, the adjusted HR for hearing dysfunction associated with maternal occupational noise exposure ≥ 85 vs. < 75 dBLAeq,8h was 1.27 . When restricted to children whose mothers worked full-time and had < 20 days leave of absence during pregnancy, the corresponding HR was 1.82 .This study showed an association between occupational noise exposure during pregnancy and hearing dysfunction in children. In view of mechanistic evidence and earlier indicative epidemiological and experimental findings, the results support that pregnant women should not be exposed to high levels of noise at work.Selander J, Albin M, Rosenhall U, Rylander L, Lewné M, Gustavsson P. 2016. Maternal occupational exposure to noise during pregnancy and hearing dysfunction in children: a nationwide prospective cohort study in Sweden. Environ Health Perspect 124:855–860; http://dx.doi.org/10.1289/ehp.1509874 A majority of women of child-bearing age in the industrialized part of the world are occupationally active today. In total, 73.7% of Swedish women of working age were employed in 2014 . This leSound is transmitted from the air over the abdominal wall and the uterus to the fetal head during pregnancy. The noise stimulates the inner ear through a soft tissue conduction route and could potentially affect the hearing of the fetus by damaging inner and outer hair cells within the cochlea, especially since the maturing cochlea is more sensitive to ototraumatic factors than the adult one . Both anThe aim of this study was to investigate if occupational exposure to noise during pregnancy is associated with an increased risk of hearing dysfunction in children in a population-based nationwide study with individual data on occupation, hearing dysfunction, and potential confounding factors.The FENIX  study is a nationwide prospective cohort study based on births in Sweden between 1986 and 2008.Information on job title and if the woman was working full time, part time, or not working at all was collected at the registration interview for prenatal care  together with background characteristics such as maternal age, smoking habits, family structure, and nationality. It was entered in the Medical Birth Register, together with information from records from the maternity wards regarding parity, gender, and date of birth. The Swedish Medical Birth Register includes 98–99% of all children born in Sweden .Information on hearing dysfunction was retrieved from the patient register, which includes diagnoses from all outpatient clinics for specialist care in Sweden from 2003 and onwards. The overall coverage in the patient register is approximately 80% , but hasData on the mothers highest completed education at the year of the child’s birth was gathered through the population-based nationwide register (LISA) holding information on all Swedish citizens at > 16 years of age .Individual information on leave of absence during each pregnancy was retrieved from the Social Insurance MIDAS database and includes information on days of sick-leave and parental leave for the time period 1986–2008. The register has a complete coverage of long-term leave of absence, since every citizen needs to register sick leave or parental leave to receive payment from the Social Insurance Agency, but it does not cover short-term sick leave (≤ 14 cohesive days). However, parental leave and special sick leave related to the pregnancy is reported from day 1 .The registers were matched with the personal identification number, unique to every Swedish citizen.Job titles were recorded at the registration interview held at the prenatal care facilities in Sweden. The occupation was recorded as free text (not coded) in the Medical Birth Register. Out of the 2,348,250 births eligible for the study (children born between 1986 and 2008 in Sweden), 1,957,189 had data on occupation. The entries were processed down into 64,398 unique occupational titles by removing characters other than letters, by harmonizing abbreviations, and correcting spelling errors. There were approximately 44,142 unique titles each held by only one mother in the cohort; these were excluded from the study because of limited resources for coding of occupations, leaving 20,256 job titles held by at least two mothers and covering 98% of the occupational titles. These were coded manually by an occupational hygienist and a safety engineer into a 6-digit AMSYK/SSYK-code based on ISCO-88   Figure . OccupatThe validated job–exposure matrix was constructed before this study and is presented in detail elsewhere . BrieflyAeq8h) of each occupation into three categories: < 75 dBA, 75–84 dBA, ≥ 85 dBA ]: other diseases of inner ear , conductive mixed with sensorineural hearing loss and sensorineural hearing loss (H90.3-7), other hearing loss , and other disorders of the ear, not elsewhere classified (including tinnitus) . This selection is based on the well-established concept that noise-induced hearing loss is of the sensorineural type, caused primarily by a cochlear damage .All analyses were performed using Cox proportional hazards regression with child’s age as the underlying time scale. Person-years for each child were calculated from the child’s birth to the age at first diagnosis or age at the end of follow-up (31 December 2008), whichever came first. Potential confounders were identified through a review of previous studies on pregnancy outcomes and occupational exposures, by observing the effect each potential confounder had on the association between exposure and outcome and by placing the potential confounders in a causal web. All variables initially assessed were selected as covariates and are presented in All analyses were restricted to single births and to employed women by excluding women who at the beginning of pregnancy  said that they were students, unemployed, housewives, refugees, or in other nonworking categories (AMSYK-code X33 and X21) during pregnancy. Women who in gestational week 10 stated an occupation that could be coded into occupational codes were included in the study. The presence of the pregnant worker at the workplace was then evaluated and certain analyses were restricted regarding work participation by combining the answers to the question on current degree  of occupational activity at beginning of pregnancy  with the registry data on days of absence  during the whole pregnancy. This was done by dividing the cohort into three categories: absent from work, part-time work, and full-time work. Absent from work included mothers who stated an occupation but also stated that they did not work at the registration interview in gestational week 10 or mothers who stated an occupation but had more than 153 days (90th percentile) of leave of absence from work during pregnancy. Part-time workers were defined as mothers who stated at the registration interview that they worked part-time or had between 20 (median) and 153 days (90th percentile) of leave of absence during pregnancy. Full-time workers were defined as mothers stating at the registration interview that they worked full-time at the beginning of pregnancy and had < 20 days (median) of sick leave during pregnancy.Statistical significance was determined by 95% confidence intervals (CIs). All analyses were performed with STATA SE 12 (StataCorp), and the study was approved by the regional ethics committee in Stockholm.The study included 1,422,333 single births. Of these, 1,320,195 had complete data for the covariates of the adjusted model. In total, 12,668 cases of hearing dysfunction were identified and included in the study; other diseases of inner ear, 133 cases , conductive mixed with sensorineural hearing loss and sensorineural hearing loss, 8,696 cases (H90.3-7), other hearing loss, 917 cases , and other disorders of the ear, not elsewhere classified (including tinnitus), 3,637 cases .Background characteristics are presented in An increased risk of hearing dysfunction among children was indicated after exposure to occupational noise during pregnancy, The risk of hearing dysfunction among children in relation to occupational noise during pregnancy, subdivided by presence at work during pregnancy, is presented in An association between maternal occupational noise exposure > 85 dBA during pregnancy and hearing dysfunction among children was indicated. The association was more pronounced when restricting the study to full-time working mothers with < 20 days’ leave of absence during pregnancy. The results are in line with two previous epidemiological cross-sectional studies on occupational noise exposure and hearing impairment that reported an increased risk of hearing impairment after exposure to high occupational noise ≥ 85 dBA during pregnancy . In addiIn the human fetus as well as in precocial mammals, the auditory system is functional well before birth . The perin utero to a large extent depends on the sound exposure outside the abdomen.The fetus is exposed to sounds not only from external sources, but also from the mother’s own body. However, the natural intrauterine sound level consist mainly of very low-frequency noise , and becin utero show a risk of adverse effects of noise on the fetal hearing during the later gestational ages, corresponding to the last trimester of pregnancy in humans  in the main analysis. The interpretation of this small increase is complex because the class is wide, with a range in exposure of 9 dBA. Most occupations are likely to be found at the lower end of the interval (75–79 dBA), and an effect in the higher end (80–84 dBA) will have a small effect on the estimated risk for the whole intermediate group. Thus, the results for the intermediate group should not be interpreted as a null result or that 75–84 dBA is a safe level for pregnant women to work in. More studies are needed before the highest safe level can be established.A wide range of hearing dysfunction diagnoses related to noise exposure was selected, mainly because noise-induced hearing dysfunctions in children are difficult to classify and thereby are sometimes classified into the selected “other” categories until a more precise diagnosis can be made. An inclusion of such a mixed group might also contribute to an inclusion of a few cases of hearing dysfunction that are unrelated to noise exposure. However, these “other” diagnoses (not including tinnitus) contributed very few cases (< 10%) and were included only in the dichotomous analyses of all hearing dysfunctions, and should therefore introduce only a minor misclassification of disease. In addition, the misclassification of hearing dysfunction in this study is most likely nondifferential, because occupational exposures to noise during pregnancy is not an established risk factor for hearing dysfunction in children, and it should therefore not make parents more prone to investigate their child’s hearing later in life.In this study, only hearing dysfunction that was sufficient to prompt an investigation by a specialist could be studied. Thus the analyses are based on few cases, but the low numbers should not be taken as evidence that the problem should be of minor public health relevance. Rather, it is possible that a larger number of cases of mild hearing dysfunction, not identified during childhood or not requiring specialist care, could be induced by occupational noise exposure. This was evident in the earlier three epidemiological studies on occupational noise during pregnancy and hearing dysfunction in children that measured hearing loss for each child by hearing thresholds instead of by diagnoses of hearing dysfunction, and thus also could detect mild hearing loss even in a small sample . Even miFuture studies should investigate the association between different noise frequency intervals and outcome measures of milder hearing dysfunction. The advice given today to pregnant women regarding occupational noise differs substantially among clinics and countries because research on this topic is lacking. This study will therefore provide important information to clinicians and policy makers and will contribute to more accurate advice and guidelines.This nationwide population-based study supports the hypothesis that occupational noise exposure during pregnancy is associated with future hearing dysfunction in children. Taken together with previous epidemiological and experimental studies as well as mechanistic data, the available data indicate that pregnant women should not be exposed to high levels of noise at work.These results need confirmation in further studies. In addition, our results indicate a need to further study the effects of intermediate levels of occupational noise, peak values, and leisure-time exposure, such as rock concerts. Although leisure-time activities are of much shorter duration, the exposure intensity may be very high.


The deep-sea hypersaline anoxic basins (DHABs) of the Mediterranean (water depth ~3500 m) are some of the most extreme oceanic habitats known. Brines of DHABs are nearly saturated with salt, leading many to suspect they are uninhabitable for eukaryotes. While diverse bacterial and protistan communities are reported from some DHAB haloclines and brines, loriciferans are the only metazoan reported to inhabit the anoxic DHAB brines. Our goal was to further investigate metazoan communities in DHAB haloclines and brines.Daptonema was the most abundant genus; three morphotypes were noted with a degree of endemicity. The majority of rRNA sequences were from planktonic taxa, suggesting that at least some individual metazoans were preserved and inactive. Nematode abundance data, in some cases determined from direct counts of sediments incubated in situ with CellTrackerTM Green, was patchy but generally indicates the highest abundances in either normoxic control samples or in upper halocline samples; nematodes were absent or very rare in lower halocline samples. Ultrastructural analysis indicates the nematodes in L’Atalante normoxic control sediments were fit, while specimens from L’Atalante upper halocline were healthy or had only recently died and those from the lower halocline had no identifiable organelles. Loriciferans, which were only rarely encountered, were found in both normoxic control samples as well as in Discovery and L’Atalante haloclines. It is not clear how a metazoan taxon could remain viable under this wide range of conditions.We report observations from sediments of three DHAB  haloclines, comparing these to observations from sediments underlying normoxic waters of typical Mediterranean salinity. Due to technical difficulties, sampling of the brines was not possible. Morphotype analysis indicates nematodes are the most abundant taxon; crustaceans, loriciferans and bryozoans were also noted. Among nematodes, We document a community of living nematodes in normoxic, normal saline deep-sea Mediterranean sediments and in the upper halocline portions of the DHABs. Occurrences of nematodes in mid-halocline and lower halocline samples did not provide compelling evidence of a living community in those zones. The possibility of a viable metazoan community in brines of DHABs is not supported by our data at this time.The online version of this article (doi:10.1186/s12915-015-0213-6) contains supplementary material, which is available to authorized users. The remarkable recent description of certain metazoans that complete a full life cycle without access to dissolved oxygen  has attrAside from the Danovaro et al.  publicatMeiofaunal metazoans , such as certain nematodes and oligochaetes, reportedly live in sediments where oxygen is undetectable e.g. , 4–6). W–6. W4–6]In addition to a complete lack of oxygen or only the presence of trace oxygen, subsurface sediments typically have high concentrations of hydrogen sulfide. Sulfide binds to cytochrome c oxidase, which is a crucial enzyme in aerobic respiration, inhibiting its normal function. In brief, this observation spawned the dogma that aerobes cannot survive long periods of exposure to sulfidic conditions. Of course, there are biochemical adaptations that permit exceptions, including the ability of some mitochondria to oxidize sulfide e.g. –9). Cert. Cert9])Herein, we report results of visually-guided sediment coring on the metazoan assemblages in DHAB halocline and adjacent normoxic, normal saline (control) sediments, providing additional information on whether observed metazoa represent living eukaryotic populations. Our attempts to sample beneath full strength brines were unsuccessful.Eastern Mediterranean DHABs, which were first described about three decades ago , are one+ concentrations in L’Atalante and Urania brines  are much higher than in the Discovery brine (68 mM) and seawater (528 mM). The highest concentration of MgCl2 found in a marine environment to date occurs in the Discovery brine  ).Diverse communities of bacteria and protists have been documented in the water columns of these basins e.g. , 22–25).–25.22–25While nematodes were the most common metazoan group present in our samples from a morphological perspective, additional metazoan morphotypes were also occasionally observed in our materials. We focus detailed discussion on the Nematoda in most subsequent sections (see below).Crustaceans, all copepods, were observed occasionally in our sediment samples Fig. . In some3 in situ seafloor volume). These individuals were obtained from both L’Atalante (n = 10) and Discovery (n = 5) lower halocline samples  (see more on this below). Rugiloricus sp. was also observed in a Discovery lower halocline sample (609c14) and S. cinziae was also observed in an L’Atalante control sample (611c3).Loriciferan specimens were only rarely observed, compared to nematodes Figs. , 4, 5, 6 cinziae , RugilorOnly two loriciferan specimens, one each from L’Atalante normoxic control and lower halocline, were detected in the Percoll density-gradient material incubated with DAPI; neither specimen had convincing DAPI label in their metazoan tissues Fig. . While nTriticella ).−2, for a mean abundance of 252 indiv. 10 cm−2. The ultrastructure of three of those bryozoans revealed that all three specimens appeared to be live at the time of fixation. Specimens had intact nuclei, mitochondria, cilia, and other organelles  labeled with CellTracker Green Fig. . Two aliMetazoans were present in many, but not all, of our DHAB sediment samples. It is important to note, however, that presence does not necessarily equate to viability. Data from our various analyses can inform on the health and activity of the metazoan communities in these DHAB environments; these discussions are presented below by higher taxonomic group.2 vs. 10–70 μM O2, respectively). In general, lower halocline sediments are expected to be anoxic while upper halocline sediments are more likely to have some dissolved oxygen. It may be relevant to note that oxygen concentration can be increased during sample handling, so detection of [O2] in a recovered pushcore from the lower halocline does not necessarily equate to detectable [O2] in situ. Furthermore, and as noted previously, the brine chemistries differ greatly from seawater as well as between the three DHABs  concentrations. Also, the density of all the brines is substantially greater than that of typical seawater. Thus, it may not be surprising to note the presence of anthropogenic items floating at the brine interface , it , it 35])Oithona) sequence were obtained from the L’Atalante upper halocline. Furthermore, small shrimp-like metazoa of unknown identity were visible via the ROV cameras when the ROV was positioned within the haloclines of all three basins (data not shown). Although it was not clear how long these organisms remained in the halocline, we speculate that they may be at least feeding within the halocline. Thus, while it is possible that copepods in DHAB halocline sediments inhabit that environment, it has yet to be demonstrated that crustaceans can complete a life cycle in anoxia. Conversely, the cyclopoid that was partially labeled with DAPI could have been a dead specimen invaded by prokaryotes or protistan scavengers, which would have also labeled with DAPI.A recent study of a shrimp from anchialine caves indicates that some crustaceans can tolerate anoxia for at least 1 week . The mosArtemia franciscana can tolerate anoxia for considerable periods, up to years [Our sequence data indicate the relatively common presence of planktonic  copepods in our sediments Table . It is lto years ; encysteIn sum, our data indicate that, while it is possible that the copepods detected in these DHABs are anaerobes adapted to the very high salt content of these brines, that possibility is not compelling without additional data.−2) occurrence of this bryozoan in a single core is not clear. While it is possible specimens existed in other samples yet were overlooked, it is also possible that the halocline transition provided a favorable environment for these meiofauna. Ctenostome bryozans are known to occur in the bathyal zone as well as in the abyss (reviewed in [The observation of a bryozoan morphotype in one of our Urania samples suggests patchy distributions of this ctenostome taxon. The significance of a rather abundant (~250 indiv. 10 cmiewed in ), but abOf all the metazoan phyla, nematodes are arguably the most likely to inhabit anoxic DHAB halocline sediments for the following four reasons: 1) nematode presence was already noted in L’Atalante DHAB sediments, although all were reportedly carcasses  nematode. (3) Som−2, with the exception of nearly 2000 nematodes 10 cm−2 in one Discovery normoxic control sample and an absence in a Urania control sample. Nematode densities in other areas of the deep Mediterranean are approximately similar to our normoxic control abundances, with means of 109.8 indiv. 10 cm−2 at a 3000-m deep station in the Algero-Provençal, 19.5 indiv. 10 cm−2 at a 3000-m deep station in the Ionian Sea, and 61.8 indiv. 10 cm−2 at a 3000-m deep station in the Levantine basin [−2 in Discovery normoxic control; 3879 and 595 indiv. 10 cm−2 in the Urania halocline transition) were much higher than those of other deep-sea sites, ranging in depth from about 1687–3000 m in the Mediterranean [Indeed, we observed dozens of nematodes from many, but not all, samples. The abundances in our normoxic normal saline controls were ~17–77 indiv. 10 cmne basin . Becauseerranean , 50 and erranean , we can In sediment samples incubated in situ with the viability indicator CellTracker Green, we observed labelled nematodes, indicating esterase activity at the time of incubation. Analysis of DAPI-labeled nematodes indicated the expected presence of nuclei throughout many specimens yet lack of copious ectobionts. Because there was no evidence for epibionts or endobionts in DAPI preparations and TEM images, we infer that the positive CellTracker Green labeling reflected nematode viability, not parasitic or scavenging prokaryotes in the body or attached to the nematode cuticle. Ultrastructural analysis revealed the presence of healthy tissue in nematodes from the L’Atalante normoxic control sample and identifiable tissues in a nematode from L’Atalante upper halocline, but no identifiable tissues in specimens recovered from the lower halocline.Daptonema-related (8 Daptonema sp. and one Zygonemella sp.) taxa found in Genbank suggests, however, that primer bias is likely not an issue. Other possible explanations for the lack of nematode sequences are that, (1) although we used a larger (8 g) than usual (~0.5–1 g) sample of sediment for RNA extraction, this amount may be insufficient due to patchy distribution of active nematodes in any sample extracted or (2) the nematode cuticle does not protect the nematode postmortem as well as the copepod exoskeleton. Additionally, signatures of pelagic copepods may have masked those of benthic nematodes merely due to abundance and rate of addition to the sediments. The deep Mediterranean is considered highly oligotrophic [Given the above rationale regarding nematode physiological ecology and the general synopsis of our nematode results, it is therefore rather surprising that there were no rRNA sequences of nematodes in our dataset – not from halocline samples or from the normoxic, normal saline control sample. This absence of nematode sequences is even more surprising given the relative abundance of pelagic copepod sequences that were obtained. The PCR primers used in this study are not known to bias against nematodes, although this possibility cannot be excluded for nematodes from such extreme habitats that may be genetically distinct. The presence of the binding sites for the primers used for all the  full-length sequences of otrophic –55, so ootrophic ), abundaDaptonema species differed among habitats and DHABs  abundances of nematodes were considerably lower in the deeper halocline samples compared to upper halocline and normoxic control samples Table , (2) theABs Fig. , we arguGiven that no identifiable nematode tissue was observed with TEM in either of the nematodes from the lower halocline samples  and since there were very low abundances of nematodes recovered from all additional lower halocline samples Table , we suggSpinoloricus cinziae, the only one described so far [Rugiloricus sp. and Pliciloricus sp., it is worth noting that a definitive match between our specimens and those reported by Danovaro et al. [Pliciloricidae. This family currently includes two genera, Pliciloricus and Rugiloricus; we used the number of plicae to affiliate our specimens to either of the two genera: about 22 plicae = Pliciloricus; 30–60 plicae = Rugiloricus [Pliciloricus  but unr3H-leucine by specimens in sediment incubations, and cellular ultrastructure analyses that show putative hydrogenosomes, which are specialized organelles heretofore unique to unicellular anaerobic eukaryotes , and enes , respectively. Importantly, one loriciferan specimen (Spinoloricus cinziae) was also obtained from a L’Atalante normoxic, normal saline control sample.Unfortunately, we did not obtain sufficient numbers of loriciferans to perform a complete suite of analyses on this intriguing taxon, including confirmation of intact and identifiable eukaryotic organs and organelles. In addition, it is noted that no rRNA sequences were obtained for loriciferans. We did document, however, that 15 loriciferans were obtained from lower halocline samples of both L’Atalante and Discovery after examining approximately 42 and 16 cmWe saw some evidence of potentially intact loriciferan tissue in some of the recovered specimens, where two Rose Bengal stained specimens each had a possible oocyte , and an ultrastructural survey would have been necessary to establish that the oocytes in our specimens were healthy.Rugiloricus sp. in both the Discovery and L’Atalante lower haloclines, the likelihood that the same species of any metazoan inhabits both the athalassohaline Discovery lower halocline and the thalassohaline L’Atalante lower halocline is low given the very different water chemistries of those two locales (Table 2+ concentration (4995 mM), which is approximately an order of magnitude higher than that of L’Atalante (410 mM); L’Atalante brine has more than 65 times more sodium than Discovery (4674 vs. 68 mM); L’Atalante brine has approximately four times more sulfate than Discovery brine ; and L’Atalante brine has more than six times greater potassium than Discovery (369 vs. 60 mM). Using that same rationale, while we found Spinoloricus cinziae in L’Atalante control and L’Atalante lower halocline samples, the likelihood that the same species of Spinoloricus inhabits both the normoxic, normal saline control sediments and thalassohaline lower halocline sediments is low due to substantial differences in the chemistry of bottom waters overlying sediments and pore waters.Although we found specimens of es Table . For exaBecause of our observations on loriciferan distribution and the visible state of specimens, we find little evidence for living loriciferan populations in the haloclines of L’Atalante and Discovery. Although we did not examine much material from Urania, that DHAB might be the most hospitable to anaerobic metazoans given the brine of Urania is less saline than the other DHABs . HoweverPontophilus), but most crustacean morphotypes and sequences were from planktonic copepod taxa, suggesting an active benthic crustacean community is of low diversity, if it exists in the haloclines. We also strove to confirm the presence of an active loriciferan community in the L’Atalante DHAB, as noted by Danovaro et al. [Our goal for this research was to investigate the presence of living metazoan communities in the DHAB haloclines and brines. Unfortunately, brine samples were not obtained. Our results suggest a community of living nematodes in normoxic, normal saline deep-sea Mediterranean sediments and in the upper halocline portions of several DHABs. There is some evidence suggesting nematode endemicity in the upper to mid-haloclines of L’Atalante and Discovery DHABs. There is also some evidence of enhanced nematode and bryozoan densities in the vicinity of the upper halocline and adjacent normoxic, normal saline regions. Occurrences of nematodes in mid-halocline and lower halocline samples did not provide compelling evidence of a living community. Crustacean occurrences in our samples suggest the possibility of living benthic taxa in the haloclines .Atlantis cruise AT18-14, sediment samples were collected using the ROV Jason from 24 November to 6 December 2011. Samples were collected in and near three DHABS: Urania, Discovery, L’Atalante (see Table www.whoi.edu/groups/DSL/). Pushcores were obtained in different zones with respect to where the halocline intersected the seafloor and salinities later confirmed by refractometer. Thus, normoxic, normal saline control samples were collected upslope from the visible manifestation of the halocline impinging the seafloor  the halocline, and reached into the halocline to sample. Minimal disturbance was observed of halocline waters (Schlieren effect) on ROV live video feed. Lower halocline cores were obtained by extending Jason’s manipulator arm into the DHAB as far as possible; when collecting cores underlying the halocline, sampling did not commence until visible disturbance (observable Schlieren) had settled. In the case of sampling sediments under the lower halocline, the conditions made it impossible to view the point at which Jason’s manipulator arm contacted the seafloor, resulting in over-penetration of some pushcores. A site was never sampled after ROV re-positioning, which introduces disturbance from the propellers.On RV http://4dgeo.whoi.edu/webdata/virtualvan/html/VV-at18-14/index.html).We do not feel that living meiofauna escaped sampling due to ROV noise or vibrations because meiobenthic organisms are less than 1 mm long, generally not very fast (e.g. ), and tyJason recovery aboard support vessel RV Atlantis, pushcores were taken within ~5 minutes to the ship’s environmental room, which was set at 9.5 (±0.5 °C), which was ~4 °C lower than in situ temperature (~14 °C). The cooler temperature was to expedite cooling of the samples, which were subject to warming on ascent and ROV recovery. Our goal was to obtain at least three pushcores  of each habitat from each DHAB; one was intended primarily for sequencing, one for quantification and one for imaging. Pushcores intended for sequencing were subcored and directly frozen, as described below. Pushcores for metazoan quantification (counts) and imaging were first sampled for overlying water to estimate salinity via a hand-held refractometer. Because of the high ionic concentrations, up to 10-fold dilution was required to register on the refractometer. The refractometer was not expected to provide precise salinity data, especially for Discovery’s very high MgCl2 brine. Rather, the data were intended as a relative indicator between samples from different halocline horizons in Discovery and L’Atalante; refractometer data was not collected for Urania pushcores. Sometimes bottom water was not available due to full penetration of the pushcore barrel; refractometer data are not reported in those cases. Halocline samples are designated in the following descending order by their relative location : upper halocline, mid-halocline, and lower halocline. After recording the refractometer reading, the surface centimeter of each pushcore was sectioned and retained. Each of the sediment samples designated for counts and imaging was preserved in either 3 % TEM-grade glutaraldehyde in a 0.1 cacodylic acid sodium salt buffer (pH 7.2) or 3.8 % formaldehyde buffered with phosphate buffered saline. Glutaraldehyde-preserved samples were intended for ultrastructural analyses with TEM (see below) and also imaging with light microscopy; formalin-preserved samples were intended for quantification and/or imaging.Upon Because pressure and temperature changes during ascent may cause metazoans to die, a subset of pushcores were incubated in situ using CellTracker™ Green , which is reported to label marine meiofauna and microbes , 10, 68 For isolation of metazoan RNA, select pushcores were subsampled with a sterile 1.4-cm inner diameter syringe corer. These subcores were frozen at −80 °C while at sea. At Woods Hole Oceanographic Institution, the cores were transferred to −20 °C for an hour and then to 4 °C for 10–15 minutes. This allowed the outer part of the subcore sediments to thaw in order to be pushed out of the syringe, but the subcores were still frozen when sectioned. The top 2 cm were placed in a sterile tube and stored frozen (−80 °C) until RNA extraction. RNA extraction, cDNA synthesis, amplification of eukaryotic small subunit rRNA genes, and subsequent analyses were identical to those described in Bernhard et al. .® density gradient extraction [® preparations, were photographed. Typically, these specimens were imaged with transmitted light (e.g. DIC or phase contrast optics) and also with epifluorescence  to image DAPI patterns, using an Olympus BX51 microscope and Olympus DP70 camera or a Zeiss AxioImager.M2 equipped with a Zeiss AxioCam MRm digital black and white camera or a Canon EOS Rebel T2i color digital camera. In cases where the metazoan was found on the blackened filter, DIC or phase contrast imaging were not possible. In cases where the sediment sample had been incubated in situ (on the seafloor) with the esterase indicator CellTracker Green (see above), digital epifluorescence images were also taken with 472-nm excitation, 520-nm emission, with either the Olympus BX51 or a Leica MZFLIII dissecting microscope.Quantitative aliquots of sediment samples were processed for metazoan enumeration and imaging, loriciferan searches, nematode morphotype identifications, and nematode and bryozoan ultrastructural analyses. Initially, all workers were blind to sample origin (i.e. habitat). However, with time, it was obvious to many which samples were from haloclines versus control (aerated) sites, due to differences in sediment color. Because the goal was to obtain as many metazoans as possible, this knowledge did not impact the outcome. Nematode and loriciferan identifications were fully blind. Many technical aliquots (portion of pushcore sample) were sieved over a 63-μm screen and the coarser fraction was examined for individuals. Also, metazoa were isolated from aliquots of sediments along with protozoa  using a traction , the supThe bryozoa and some loriciferans were also imaged using a Nikon Eclips 90i microscope fitted with DIC optics and a DS-5 M Nikon digital camera driven by Nikon ACT-2U software v.1.4.Because a portion of our samples were sieved using a 63-μm screen, which may be considered large for deep-sea nematode isolation , our nem® density-gradient centrifugation; the supernatant was stained with Rose Bengal and metazoan meiofauna were sorted under a stereomicroscope and identified to coarse taxonomic level [Aliquots of select aldehyde-preserved samples were sent to the Marine Biology Research Group of Ghent University for nematode morphotype analyses. The >32-μm fraction of sediments was subject to a Ludoxic level . NematodAs noted, one-centimeter sections of sediment pushcores were fixed using TEM fixative . In the laboratory, nematode specimens were isolated from the >63-μm sediment fraction and embedded for TEM as per standard protocols (e.g. ). Specimhttp://www.bco-dmo.org/project/2177 and http://www.bco-dmo.org/project/2134), and on the ROV Jason Virtual Van website . Supplemental images of loriciferans can be found at the Woods Hole Open Access Server (WHOAS) at http://hdl.handle.net/1912/7550. Sequence data is available at https://trace.ddbj.nig.ac.jp/DRASearch/study?acc=SRP049010 and a mapping file showing how to link sequence identifiers with environmental source (basin and habitat) in Additional file Cruise data is available at the Biological and Chemical Oceanography Data Management Office (BCO-DMO) web site (


Acute Myeloid Leukemia (AML) is characterized by various cytogenetic and molecular abnormalities. Detection of these abnormalities is important in the risk-classification of patients but requires laborious experimentation. Various studies showed that gene expression profiles (GEP), and the gene signatures derived from GEP, can be used for the prediction of subtypes in AML. Similarly, successful prediction was also achieved by exploiting DNA-methylation profiles (DMP). There are, however, no studies that compared classification accuracy and performance between GEP and DMP, neither are there studies that integrated both types of data to determine whether predictive power can be improved.Here, we used 344 well-characterized AML samples for which both gene expression and DNA-methylation profiles are available. We created three different classification strategies including early, late and no integration of these datasets and used them to predict AML subtypes using a logistic regression model with Lasso regularization.We illustrate that both gene expression and DNA-methylation profiles contain distinct patterns that contribute to discriminating AML subtypes and that an integration strategy can exploit these patterns to achieve synergy between both data types. We show that concatenation of features from both data sets, i.e. early integration, improves the predictive power compared to classifiers trained on GEP or DMP alone. A more sophisticated strategy, i.e. the late integration strategy, employs a two-layer classifier which outperforms the early integration strategy.We demonstrate that prediction of known cytogenetic and molecular abnormalities in AML can be further improved by integrating GEP and DMP profiles. FLT3), nucleophosmin (NPM1 ) or CCAAT enhancer binding protein alpha (CEBPA), are nowadays used for risk-classification of patients [double-mutant CEBPA[FLT3, NPM1, or with certain chromosomal abnormalities  [Over the last decade, microarray technologies that measures gene expression profiles (GEP) proved to be effective for the detection of biomarkers for diagnosis and prognosis of disease and for helping with the determination of drug treatment . More repatients ,5. Currepatients . Alternae-mutant , challen 5q, 7q) , where iNPM1 mutations are detected [CEBPA, which is an important transcription factor in AML and a mutation (e.g. double mutation in CEBPA) can cause a selective block in differentiation, a hallmark of AML [double-mutant CEBPAcases, but do not harbour this mutation. Follow-up experimentation showed that these patients contained a unique epigenetic feature, i.e. CEBPA is silenced by DNA hypermethylation (denoted as the silenced CEBPAsubtype) [Besides gene expression profiles, DNA-methylation profiles (DMP) also provide insight into the pathology of Acute Myeloid Leukemia (AML) . In partdetected . This suk of AML ,11. Howesubtype) . Thus, bThe combination of GEP with other data types measured in the same sample is not frequently available. To derive more reliable and robust gene signatures and improve classification performance many methods have therefore been developed that integrate GEP with prior knowledge. Some examples include integration with protein-protein networks ,14, protWe hypothesize that the prediction of AML subtypes can be improved by combining gene expression and DNA-methylation profiles, as it has previously been shown that DNA-methytlation patterns contain complementary information on top of the gene expression patterns in an unsupervised analysis. For example, it has been shown that integrating GEP and DMP revealed two unique, and clinically relevant AML patient-clusters that could not be discovered when using either GEP or DMP alone .While both gene expression and DNA-methylation profiles have been used to gain insight in AML ,9, theredouble-mutationCEBPA, and silencedCEBPA. However, the accuracy  and classification performance , double-mutation CEBPA, and silenced CEBPA. This trend is also apparent for the other subtypes  or DMP alone (DMP P= 1.5E - 04). Nevertheless, the classification accuracy was equal for all integration strategies (F - score = 0.667).The early integration strategy improves  the classification accuracy for all 15 subtypes compared to DMP, and for 8 out of 15 subtypes for GEP . Interestingly, the selected features obtained with the early integration strategy are composed of both GEP and DMP features .In terms of number of features selected by the classifier, we can read from Figure To summarize, an early integration strategy can improve subtype classification. However, for some subtypes the classification performance is equal to that obtained using GEP or DMP alone. The latter observation may be explained because no complementary information is carried in the DNA-methylation and gene expression profiles for certain subtypes. If this is the case, early integration may even result in loss of performance as the dimensionality of the original feature space approximately doubles in size. As a result, classification will suffer more from the small sample size problem.F - score = 0.8) compared to the late integration (F - score = 0.77). The better accuracy of the late integration can be explained in two ways: first, in the early integration strategy we standardized all features to the same scale to make them equally important, whereas in the late integration no additional normalization is required for GEP and DMP as we train the 2nd layer classifier on the outputs of the GEP and DMP regressors. Secondly, the threshold on the posterior probability is not fixed in the late integration scheme but learned by the nearest mean classifier (NMC). The latter is illustrated in Figure AUC = 0.799, F - score = 0.68) compared to GEP , DMP  individually or early integration   showed better accuracy in the early integration  Figure  and 1B. ITDFLT3/wt NPM1, t or inv(16). Nevertheless, the global test showed in these subtypes that the features in an early integration strategy were more indicative for the subtype compared to DMP, underpinning the observation that gene expression and methylation are complementary measurements when subtyping leukemia patients.NRAS, TKDFLT3, 3q, 7q and 11q23 abnormalities showed improved prediction using the late integration strategy but nevertheless, remain difficult to predict and therefore still require alternative methods. This may be due to the high variability of expression or methylation profiles within the subtype, consequently features based on GEP or DMP cannot accurately characterize the subtype. In fact, the test statistic from the global test indeed indicates no significant association with 3q and 7q patients by using the features from either the GEP (P = 0.122 and P = 0.051 respectively) or DMP (P = 0.173 and P = 0.06 respectively). Perhaps for these cases, a multitude of other complementary data sets, such as microRNA, Copy Number Variation (CNV) or pathway information, is necessary to delineate these subtypes.Patients with In this study we have chosen to build a classifier that is optimized for predictive power and the derived signatures may therefore not be appropriate to study the biological effect. We demonstrate a supervised approach that can improve the predictive power for each cytogenetic and molecular abnormality by employing an early or late integration strategy, whereas the late integration strategy is recommended above early integration.GEP N= 21678 refseqs). For the same set of samples, genome-wide DNA-methylation data (DMP) was measured with the HELP-assay [DMP N= 22725 features). Both datasets are annotated using UCSC hg19 genome build, and are available from the NCBI Gene Expression Omnibus accession numbers GSE14468 (HOVON-SAKK cohort) and GSE18700, respectively.For 344 adults, clinical, cytogenetical and molecular characteristics were analysed using bone marrow or peripheral blood, as described previously . For eacLP-assay  and pre-LP-assay  , i.e. 3q, 7q, and 11q23 cases. We used the 15 subtypes as classification labels. Note that samples can contain multiple abnormalities.Groups of AML patients that are characterized by common cytogenetic or molecular abnormality are denoted as subtypes. We studied fifteen of the most common subtypes, which can roughly be categorized into three risk groups . AML subtypes in the good risk group are inv(16), t, t. The intermediate risk group contains patients with molecular abnormalities, i.e. The AML subtypes are classified using three different strategies: i) no integration using the GEP or DMP-dataset individually); ii) early integration; and iii) late integration Figure . For eacFor the GEP and the DMP input datasets we followed the DLCV scheme, for each of the datasets separately. The optimal set of features, i.e. those discriminating between patients from one subtype and the remaining patients, were determined in the DLCV inner loop. Subsequently, we used this set of features to classify samples in the independent validation set (DLCV outer loop) and then calculate the classification performance and accuracy for each data type.TOTAL N= 44403 features. Then, we followed the DLCV scheme, with the exception that we now provided the regression model with all features.In this strategy, we combined all the features by concatenating the GEP and DMP features yielding For the late integration strategy, we established a two-layer classifier Figure . InitialThe F-score is used to test the prediction accuracy, which considers both the positive predictive value (precision) and the true positive rate , and varies between 0 (worst accuracy) to 1 (best accuracy). To assign a sample to a class, we used the default threshold of 0.5 on the posterior probability obtained from the logistic regressor. As a result, samples for which the posterior probability is between 0.5 and 1.0 are assigned to the subtype of interest. The F-score is especially of interest in diagnostic settings where it is important to know how many patients (samples) are correctly or wrongly classified.The Area under the curve (AUC) is computed on the Receiver Operator Characteristics (ROC), which integrates performance scores over all possible thresholds on the posterior probability obtained from the regressor. This effectively considers all possible assignments of samples to the subtype by the regressor.P-value based on the null hypothesis that there is no information in the given input features related to the sample label (e.g. subtype). In fact, the global test method applies a regression model to test the null hypotheses that the variance of regression coefficients of all input features is zero and subsequently calculates a test statistics.To examine whether the global pattern of an input set significantly associates with the subtype, we apply the global test method . The useThe authors declare that they have no competing interests.ET and SB analysed and interpreted the data, and drafted the manuscript. ET, SB, MJTR, and JdR contributed to design of the study, the discussions and editing of the manuscript. All authors provided relevant input at different stages of the project, and read and approved the final manuscript.Table S1. Table containing the classification performance for different integration strategies.Click here for file


Synapses between cochlear nerve terminals and hair cells are the most vulnerable elements in the inner ear in both noise-induced and age-related hearing loss, and this neuropathy is exacerbated in the absence of efferent feedback from the olivocochlear bundle. If age-related loss is dominated by a lifetime of exposure to environmental sounds, reduction of acoustic drive to the inner ear might improve cochlear preservation throughout life. To test this, we removed the tympanic membrane unilaterally in one group of young adult mice, removed the olivocochlear bundle in another group and compared their cochlear function and innervation to age-matched controls one year later. Results showed that tympanic membrane removal, and the associated threshold elevation, was counterproductive: cochlear efferent innervation was dramatically reduced, especially the lateral olivocochlear terminals to the inner hair cell area, and there was a corresponding reduction in the number of cochlear nerve synapses. This loss led to a decrease in the amplitude of the suprathreshold cochlear neural responses. Similar results were seen in two cases with conductive hearing loss due to chronic otitis media. Outer hair cell death was increased only in ears lacking medial olivocochlear innervation following olivocochlear bundle cuts. Results suggest the novel ideas that 1) the olivocochlear efferent pathway has a dramatic use-dependent plasticity even in the adult ear and 2) a component of the lingering auditory processing disorder seen in humans after persistent middle-ear infections is cochlear in origin. Age-related hearing loss is the most common cause of hearing impairment in adults. Loss of threshold sensitivity tends to be bilaterally symmetric, more pronounced at high frequencies, and associated with difficulty in speech discrimination, causing social isolation and cognitive deficits –3. WhileLoss of cochlear hair cells is a major cause of threshold elevation in age-related hearing loss. In both humans and animals, aging ears show progressive hair cell loss, beginning in the basal, high-frequency end of the cochlear spiral. However, hair cells are not the most vulnerable elements. In both noise-induced  and age-In aging mice, the number of synaptic connections between cochlear sensory neurons and hair cells decreases by 50% over the 2-year lifespan, and by 25% at middle age, when there is not yet any loss of hair cells . This agHearing thresholds in some non-industrialized societies show minimal deterioration with age, suggesting that cochlear aging is exacerbated by a lifetime of acoustic insults –18. InspControl animals (n = 11); 2) OCx animals (n = 16), in which the olivocochlear (OC) bundle was surgically transected unilaterally and 3) TMx animals (n = 10), in which the tympanic membrane (TM) was removed unilaterally. Two additional mice from the OCx group were analyzed separately, as they had conductive hearing loss due to otitis media (OM) on the side contralateral to the OC transection.Male CBA/CaJ mice at 6 wks of age were assigned to one of four groups: 1) OCx group, the mouse was held in a stereotaxic apparatus with the scalp retracted. A #11 scalpel blade was lowered into the brain through a skull opening at a position 5.9 mm caudal and 1 mm lateral to the bregma and to a depth of 6.9 mm from the surface. For the TMx group, the entire pars tensa of the TM was resected using a right-angle hook. The status of the membrane was re-checked at each cochlear function test day (see below); the resection sometimes had to be repeated, but always became permanent by 16 wks.For all groups, mice were anesthetized with xylazine  and ketamine . For the After surgery, all mice were returned to the animal care facility. Ambient sound pressure levels inside the cages were previously described . When anCochlear function was assessed bilaterally via auditory brainstem responses (ABRs) and distortion product otoacoustic emissions (DPOAEs) at 8, 11, 16, 22, 32, 45 and 64 wks of age. After the final cochlear function test, cochleas were fixed by intracardiac perfusion and removed for histological processing and confocal analysis of hair cell and synaptic degeneration.All procedures were approved by the IACUC of the Massachusetts Eye and Ear Infirmary. Two-way repeated-measure ANOVAs, adjusted with the Holm-Bonferroni correction, were used to assess the significance of group differences.For measuring ABRs and DPOAEs, animals were anesthetized with xylazine  and ketamine  and placed in an acoustically and electrically shielded room maintained at 32°C. The intertragal notch was slit prior to each electrophysiological recording session to allow stereotyped seating of the acoustic delivery system with a direct and unobstructed path to the eardrum. Acoustic stimuli were delivered through a custom acoustic system consisting of two miniature dynamic earphones used as sound sources (CUI CDMG15008-03A) and an electret condenser microphone (Knowles FG-23329-PO7) coupled to a probe tube to measure sound pressure near the eardrum.2 rise-fall) delivered in alternating polarity at 35/sec. Electrical responses were measured via Grass needle electrodes at the vertex and pinna with a ground reference near the tail and amplified 10,000X with a 0.3–3 kHz passband. Responses to as many as 1024 stimuli were averaged at each sound pressure level, as level was varied in 5 dB steps from below threshold up to 80 dB SPL. For DPOAEs, stimuli were two primary tones f1 and f2 (f2/f1 = 1.2), with f1 level always 10 dB above f2 level. Primaries were swept in 5 dB steps from 20 to 80 dB SPL (for f2). The DPOAE at 2f1-f2 was extracted from the ear canal sound pressure after both waveform and spectral averaging. Noise floor was defined as the average of 6 spectral points below, and 6 above, the 2f1-f2 point. Threshold was computed by interpolation as the primary level (f2) required to produce a DPOAE of 0 dB SPL.Digital stimulus generation and response processing were handled by digital I-O boards from National Instruments driven by custom LabVIEW software. For ABRs, stimuli were 5-msec tone pips  with 0.3% Triton X-100 (TX) in PBS for 1 hr, followed by incubation for ~19 hrs at 37°C in primary antibodies diluted in 1% NHS with 0.3% TritonX. Primary antibodies included 1) mouse (IgG1) anti-CtBP2 from BD Biosciences at 1:200 to visualize pre-synaptic ribbons, 2) mouse (IgG2a) anti-GluA2, from Millipore at 1:2000 to visualize post-synaptic glutamate receptor patches, and 3) rabbit anti-VAT from Abcam at 1:200 to allow quantification cochlear efferent terminals. Primary incubations were followed by 2 sequential 60-min incubations at 37°C in species-appropriate secondary antibodies with 0.3% TritonX.Histological analyses were based on high-power confocal z-stacks of the sensory epithelium whole-mounts obtained at half-octave intervals along the cochlear spiral from 5.6 to 64 kHz. To accurately identify regions of interest, cochlear lengths were obtained for each case by tracing the cochlear spiral in low-power images of the dissected pieces using a custom plug-in running under ImageJ (v1.44) that translates cochlear position into frequency using the map for the mouse . ConfocaFour types of information were extracted from inner hair cell and outer hair cell areas in each cochlea: 1) counts of afferent synapses, 2) spatial analysis of afferent synapse locations and inner hair cell alignment along the modiolar-pillar axis, 3) counts of hair cell survival, and 4) the degree of de-efferentation in inner and outer hair cell areas.connected components tool in Amira® (Visage Imaging), which finds and displays each voxel space in an image stack containing pixel values greater than a user-set criterion. To quantitatively assess the pairing of pre- and post-synaptic elements, we used custom software that extracts the voxel space within 1 μm around each ribbon (or receptor patch) and produces a thumbnail array of these miniature projections, that can be scanned to count synapses (i.e. ribbons with closely apposed receptor patches) vs. orphan ribbons or orphan receptor patches [Pre-synaptic ribbons and post-synaptic glutamate receptor patches in the inner hair cell area were counted from each confocal z-stack using the  patches . The synTo assess the spatial organization of inner hair cell afferent synapses, and the alignment of inner hair cells themselves, we used an approach described in detail elsewhere . BrieflyHair cells in each z-stack were counted by increasing the image output-gain (gamma adjust): Inner hair cell nuclei stain faintly with the CtBP2 antibody, and the outer hair cell somata are visible via their faint background label in several confocal channels, as well as by the presence of synaptic ribbons, even when the efferent terminals are missing.The degree of de-efferentation was assessed in both inner and outer hair cell areas from maximum projections of the VAT-immunostaining in the z-stacks. In both inner and outer hair cell area, the OC innervation was quantified by applying the same threshold algorithm from ImageJ and counting the total number of pixels in maximum projections.In one group of young (6 wk) mice, we removed most of the TM on one side, and in a second group, we cut the OC bundle on one side . In a thConductive hearing loss was produced by removing the tympanic membrane (TM). The sound attenuation produced by TM perforation depends strongly on the size of the defect –23. HereOCx ears, age-related threshold deterioration was amplified on the side ipsilateral to the cut, as expected based on prior work showing that cochlear de-efferentation exacerbates both cochlear synaptopathy and hair cell damage [TMx ears, rather than being protected from age-related threshold changes, also showed increased threshold deterioration, especially in the ABR measures, although with a delayed onset relative to the OCx ears  lifespan , and cocl damage . To our OCx ears . In the OCx cases, otitis media (OM) developed immediately in the ear opposite the OC lesion, and never resolved. The TM opacity associated with otitis media is clearly visible during cochlear function testing. As seen in In two of the OCx ears, ABR threshold shifts in the ipsilateral ears were flatter with frequency .At 64 wks, the ears with conductive hearing loss showed slightly more ABR threshold elevation at high frequencies (50–60 dB) than at low frequencies (35–45 dB), compared to age-matched controls . In OCx requency . At high removal ; howeverCx cases , especiaIn control ears, there is virtually no loss of either inner or outer hair cells over the first 64 wks of life . RemovalTo evaluate the OC innervation, we immunostained the cochlear sensory epithelium with a cholinergic marker: vesicular acetylcholine transporter (VAT). As seen in confocal image stacks, each outer hair cell is normally contacted by a cluster of VAT-positive MOC terminals . In the OCx cases. As shown in When accurately positioned, brainstem cuts  can compTMx cases also showed significant ipsilateral decrease in LOC innervation density , despite MOC loss approaching 40% in some cochlear regions  synapse on the side of the inner hair cell closer to the modiolus, while low-threshold fibers with high SRs synapse on the side of the hair cell closer to the pillar cells . Since lAs seen in In prior work , we noteper se, was the common cause of the cochlear synaptopathy  LOC and the (myelinated) MOC fibers run intermingled within the OC bundle, surgical transection typically removes both types of efferent fibers in roughly equal proportion. Chronic conductive hearing loss, in contrast, affected only the density of LOC terminals . Since TData obtained in this, and a prior, study suggest that LOC-mediated synaptic protection requires a “threshold level” of roughly 50% of the normal innervation density, above which, the synapses are protected, and below which some population of cochlear nerve synaptic terminals is at risk. In the present study, TM removal and OC bundle transection caused similar degrees of cochlear synaptopathy in the basal half of the cochlea , yet theThe loss of LOC innervation was also correlated with hypotrophy of the inner hair cells, manifested as an increased alignment of adjacent nuclei when viewed along the cochlea’s spiral axis . AlthougMOC terminals are virtually all cholinergic –39, and Sensory deprivation has long-lasting, deleterious effects on brain and behavior. In studies of the auditory system, disruption of the middle-ear has been a common manipulation used to acoustically deprive one ear. Most such studies have imposed the conductive hearing loss during the neonatal period –46 and mHere, we introduced acoustic deprivation at 6 wks of age, a time in the mouse when the ear appears mature in form, as seen in histological analysis at the light microscopic level, and in function, at least as far as can be observed by measuring thresholds by ABRs and DPOAEs . StudiesIn prior work, we suggested that one function of the LOC system was to binaurally balance excitability of the left and right cochlear nerves in the face of changing sound transmission, due, for example to a unilateral conductive hearing loss . Such biper se, damages the efferent and afferent innervation of the hair cells, in ways similar to that seen in age-related and noise-induced hearing loss. Although the mechanisms underlying cochlear de-efferentation following sound-deprivation are not known, its effects need to be considered in the management of chronic conductive hearing loss in clinic.At least 80% of children will experience one or more bouts of otitis media before they reach 3 years of age, making it the most common cause for physician visits and medication prescriptions among children in the USA . These b


Background noise can make speech communication tiring and cognitively taxing, especially for individuals with hearing impairment. It is now well established that better working memory capacity is associated with better ability to understand speech under adverse conditions as well as better ability to benefit from the advanced signal processing in modern hearing aids. Recent work has shown that although such processing cannot overcome hearing handicap, it can increase cognitive spare capacity, that is, the ability to engage in higher level processing of speech. This paper surveys recent work on cognitive spare capacity and suggests new avenues of investigation. Speech is the main mode of communication for most people. If speech understanding is compromised by noise or hearing impairment, communication may become harder, leading to limitations in social participation. Technical compensation is available in the form of hearing aids. However, although the amplification provided by hearing aids can improve speech understanding in quiet, persons with hearing impairment still have disproportionately large difficulties understanding speech in noise. One of the reasons for this may be that when the cognitive resources required for speech comprehension are engaged in the lower level processes of deciphering the signal, fewer resources may be available for higher level language processing. In other words, cognitive spare capacity is reduced.Speech comprehension requires the auditory ability to hear the signal and the cognitive ability to relate this information to the existing knowledge stored in semantic long-term memory , 2. The Speech processing engages a clearly defined cortical network involving the classical language areas in the left inferior frontal cortex and superior temporal gyrus , 4. The Around 25% of the population in developed countries has a hearing impairment severe enough to interfere with speech communication . HearingTypes of hearing loss are traditionally categorized according to site of lesion: impairment of sound transmission in the external or middle ear is referred to as conductive hearing loss, while other types of hearing loss are referred to as sensorineural. Sensorineural hearing loss can be further subdivided into sensory loss, resulting from impairment of cochlear function, retrocochlear loss, resulting from impairments relating to conduction in the auditory nerve or brainstem, and central losses, resulting from impairments in cortical processing of the auditory signal. Sensorineural hearing loss is the major diagnostic category and includes age-related hearing loss or presbyacusis. These categories are relatively coarse and it has been suggested that they may be inadequate for pinpointing the contribution of hearing loss to communication difficulties under adverse listening conditions .The primary diagnostic tool in audiology is the pure tone audiogram. This method of determining frequency-specific hearing thresholds is based on delivering sine waves of different intensities to each ear and asking the patients to respond by pressing a button each time they hear a sound. The resulting resolution is poor, and since this procedure requires the processes of intention and attention that characterize listening as opposed to simply hearing and thus tap into cognitive processes that may also be declining with age, diagnosis may be confounded. Other diagnostic tools include measures of auditory brainstem response and otoacoustic emissions which may be more independent of high-level cognitive contribution, although it has recently been shown that cognitive load influences brainstem responses  and otoaThe most important objective for hearing aid signal processing is to make speech audible . This isAcoustic noise impacting speech perception can be categorized as signal degradation, energetic masking, and informational masking . Signal Persons with hearing impairment have particular difficulties listening in noise which may be reflected in recruitment of neural networks supporting compensatory processing , 36 wherCognitive processes are required to focus on the speech signal and match its contents to stored knowledge , 2. WhenThe ELU model  links inMore than a decade ago, it was established that there is a relation between cognitive ability, in particular WM capacity, and the benefit obtained from hearing aid signal processing , 67–69. The capacity of WM can be increased by training, suggesting an inherent plasticity in the system , 76. TraWhen listening takes place in adverse conditions, it is clear that the cognitive resources available for higher level processing of speech will be reduced . In otheCSC is closely related to WM in that it is concerned with short-term maintenance and processing of information . Work toRecent work has shown that noise reduction (NR) in hearing aids can enhance CSC by improving retention of heard speech , 87. ThiSpeech communication under adverse conditions is likely to draw on cognitive functions other than simply memory retention , 59. In Across three different studies including persons with and without hearing loss, an interesting pattern of results has emerged , 84, 93.Adults with normal hearing who perform CSCT in noisy conditions do not show this pattern  and nor The ELU model describes the way in which the mapping of phonological structure of target speech onto phonological representations in the mental lexicon  is mediaLexical access is faster when phonological representations are easier to distinguish from each other , 104. HoIn summary, phonological structure of target speech material is not only influenced by speaker characteristics but also by distortion due to hearing aid signal processing. Phonological representations in the mental lexicon may be influenced by long-term effects of both hearing impairment and signal processing. Further, both of these may have distinct neural signatures. Measures designed to improve phonological distinctiveness of both target speech and phonological representations are likely to enhance CSC and support speech communication under adverse conditions. This deserves further investigation.Provision of semantic context can facilitate speech understanding under adverse conditions. This process engages language networks in left posterior inferior temporal cortex and inferior frontal gyri bilaterally . StudiesText cues can facilitate speech understanding in noise when they match the semantic content of the auditory signal –116 and Sensory and cognitive functions decline with age , 119. SeRecent work has shown that older adults show less activation in auditory cortex than younger adults while listening to speech in noise, especially at poor signal to noise ratios and compensate by recruiting prefrontal and parietal areas associated with WM . EpidemiSpeech communication in adverse conditions makes specific demands on cognitive resources. In particular, WM capacity and executive function are engaged in unravelling the speech signal. This depletes CSC and leaving fewer resources for higher level processing of speech. CSC is influenced by cognitive load, noise, visual cues, and aging and can be enhanced by appropriate hearing aid signal processing. The phonological structure and semantic content of speech influence processing mechanisms and engagement of cognitive resources. Optimizing CSC is an important aim for preserving speech communication into old age. We have reviewed evidence suggesting that CSC may be enhanced by a number of means including cognitive training and providing the optimal balance between visual, phonological, and semantic information. Future research should focus on finding ways to optimize CSC.


Streptococcus dysgalactiae subsp. equisimilis (SDSE) is a human pathogen that colonizes the skin or throat, and causes a range of diseases from relatively benign pharyngitis to potentially fatal invasive diseases. While not as virulent as the close relative Streptococcus pyogenes the two share a number of virulence factors and are known to coexist in a human host. Both pre- and post-genomic studies have revealed that horizontal gene transfer (HGT) and recombination occurs between these two organisms and plays a major role in shaping the population structure of SDSE. This review summarizes our current knowledge of HGT and recombination in the evolution of SDSE. Streptococcus pyogenes  and Streptococcus dysgalactiae subsp. equisimilis  are the two most common β-hemolytic streptococci recovered from humans. Along with Streptococcus canis, a bacterium causing infections in dogs and sometime humans  is associated with changes in virulence of some lineages  in SDSE. However, the emm-gene is not part of this island, and is located in a distant part of the chromosome  locus. The locus also includes genes that encode a transcriptional regulator, pilus-associated proteins and other extracellular matrix binding proteins (skg) is another SDSE gene with a mosaic structure. Streptokinase interacts with plasminogen ultimately resulting in the formation of the serine protease plasmin. Plasmin activity subsequently assists the dissemination of streptococci  transporter  and topoisomerase IV (parC). Resistance to fluoroquinolones has been reported in several streptococcal species , glutamine transport protein (gtr), glutamate racemase (murI), DNA mismatch repair protein (mutS), transketolase (recP), xanthine phosphoribosyltransferase (xpt), and acetoacetyl-coathioloase (atoB). With the exception of atoB the genes used for MLST in SDSE are the same as used for the GAS MLST scheme. Analogous to the HGT involving emm-genes the presence of GAS MLST alleles in SDSE provides strong evidence for the transfer of these theoretically fixed non-MGE-related genes , has also been identified in SDSE  in SDSE was described by cus suis , 2012 anDSE 5580 . These It groups . Based o in SDSE . RD2 wasalactiae . RD2 carOur knowledge of the population structure, molecular epidemiology and pathogenic mechanisms of SDSE are lacking when compared to GAS and other pathogenic streptococci. However, it is apparent that HGT and recombination play an important role in generating genetic diversity within the species. New phenotypic repertoires associated with HGT may occasionally give rise to clones with increased fitness that could conceivably become dominant strains, or have increased virulence. The scale of HGT varies from replacement of individual domains within a gene to large MGE-associated transfers. While both bacteriophage and ICEs contribute to genetic diversity, current evidence indicates that it is ICEs that provide most novel genes to the population. In the future, comparative genomic sequencing of large numbers of SDSE isolates will provide a much greater depth to our understanding of these events, and how this bacterium has, and continues to evolve.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Recent investigations have identified that commercially available dietary supplements, containing a combination of thermogenic ingredients, can increase resting metabolic rate (RMR). Thermogenic dietary supplements can have a positive influence on RMR, but the magnitude can vary based on the active ingredient and/or combination of active ingredients. Additionally, further safety evaluation is needed on multi-ingredient supplements that contain caffeine, due to its potential effect on heart rate (HR) and blood pressure (BP). The purpose of this study was to examine the effects of a commercially available dietary supplement on RMR and hemodynamic variables in healthy females.13 female participants  volunteered to participate in this investigation. Participants underwent two testing sessions separated by approximately 7 days. On their first visit, participants arrived to the laboratory after an overnight fast and underwent a baseline RMR, HR, and BP assessment. Next, each participant ingested a thermogenic dietary supplement or placebo and repeated the RMR, HR, and BP assessments at 60, 120, and 180-minutes post-ingestion. Approximately 1-week later, the alternative supplement was ingested and the assessments were repeated in the exact same manner. Data were analyzed via a 2-factor [2x4] within-subjects repeated measures analysis of variance (ANOVA). Post-hoc tests were analyzed via paired samples t-tests.p < 0.05) as compared to baseline RMR values. No changes in RMR were observed for the placebo treatment (p > 0.05). Heart rate was not significantly affected at any time point with either supplement; however, main effects of treatment and time were observed for both systolic and diastolic blood pressure (p < 0.05).Repeated measures ANOVA revealed a significant effect for time relative to raw RMR data. Post-hoc analysis revealed that the dietary supplement treatment significantly increased RMR at 60-minutes, 120-minutes, and 180-minutes post ingestion (The thermogenic dietary supplement treatment experienced greater elevations in RMR as compared to baseline. Due to the slight elevations in blood pressure, caution should be taken for those with increased risk for hypertension or pre-hypertension. Taken on a daily basis, thermogenic dietary supplementation may increase overall energy expenditure, potentially leading to reductions in fat mass over time. Dietary supplements containing thermogenic ingredients are commonly used amongst fitness-minded individuals in an attempt to increase resting metabolic rate (RMR) and facilitate fat loss. Recent investigations have identified that commercially available thermogenic supplements and ingredients typically contained in such products can increase RMR in healthy subjects –3, and wCaffeine supplementation has previously been shown to enhance lipolysis and fat oxidation . HoweverIt is generally accepted that caffeine increases resting energy expenditure (REE) through activation of β2 and β3 adrenergic receptors, as well as activation of cyclic AMP (cAMP) , 13, cauThere is some concern that stimulant-based thermogenic supplements may adversely affect hemodynamic variables, such as heart rate (HR) and blood pressure (BP). Some trials have shown acute increases in HR and BP following ingestion of thermogenic supplements containing caffeine plus ephedra , 27. OthWhile it is known that these ingredients can have a positive influence on RMR, the magnitude can vary based on the active ingredient and/or combination of active ingredients. Additionally, further safety evaluation is needed on multi-ingredient supplements that contain caffeine because of its potential effect on HR and BP. Therefore, the primary objective for this study was to determine the effects of a thermogenic dietary supplement on RMR in female participants. A secondary objective of this study was to determine the effects of the thermogenic dietary supplement on resting HR and BP.Thirteen healthy females  between the ages of 18 and 50 years volunteered to participate in this randomized, double-blind, placebo controlled, cross-over study. All participants reported engaging in resistance exercise and endurance exercise most of days of the week. The research protocol was approved by the University of South Florida Institutional Review Board. Following an explanation of all risks and benefits associated with the experimental protocol, each participant gave her informed consent to participate in this study. Participants were screened for participation based on established criteria set forth by the American College of Sports Medicine . In ordeThe study utilized a randomized, double-blind, placebo-controlled, cross-over design. Participants reported to the Performance and Physique Enhancement Laboratory following an overnight fast (a minimum of an 8-hour fast) and a 24-hour avoidance of exercise on two occasions separated by at least 24 h. The laboratory was climate controlled with the average temperature, humidity, and barometric pressure over all testing sessions being 21.4 °C, 47 %, and 762 mmHg, respectively. After arriving to the laboratory, a coin was flipped to randomly determine the order of the dietary supplement ingestion. If the participant were randomized to ingest the thermogenic dietary supplement on the first testing session, they would ingest the alternate treatment (placebo) on the second and final testing session. Likewise, if the participant were randomized to ingest the placebo treatment on the first testing session, they would ingest the alternate treatment on the subsequent laboratory visit. Testing sessions for both laboratory visits occurred between the hours of 5:30 am and 9:30 am, with the majority of all assessments beginning at 7 am.Upon arriving to the laboratory, participants were encouraged to use the restroom to empty their bladders. Next, the participant had their body weight measured on a physician beam scale  and then sat in a reclined position with their feet elevated for a 5-minute period. After sitting quietly for 5 min, participants had their resting heart rate and blood pressure recorded using an automated, oscillometric blood pressure monitor . This method of automated, oscillometric blood pressure measurement has been validated in the scientific literature . Heart rr = 0.97, p = 0.579)  within-subjects repeated measures analysis of variance (ANOVA) using SPSS version 22.0. Post-hoc tests were analyzed via paired samples t-tests. In addition, baseline RMR data for the two separate testing days was compared via a paired samples s et al. . A pairet-test revealed no significant difference in baseline RMR between the two treatments. Repeated measures ANOVA revealed a significant treatment x time  and main effect for time  relative to raw RMR data, but no difference in the main effect for treatment  was observed. Post-hoc analysis revealed that the thermogenic dietary supplement treatment significantly increased RMR at 60 min, 120 min, and 180 min post ingestion , as compared to baseline RMR values  was observed between the thermogenic supplement and placebo treatments . For systolic blood pressure (SBP), no treatment x time interaction effect (p = 0.748) was observed, but a main effect for treatment (p = 0.031) and main effect for time (p = 0.005) was revealed. For diastolic blood pressure (DBP), a treatment x time interaction effect (p = 0.028), a main effect for treatment (p = 0.005), and a main effect for time (p = 0.009) were observed. Raw data for RMR and hemodynamic variables is summarized in Table Heart rate was not significantly affected at any time point with either supplement  increase in RMR, regardless of the timing of the menstrual cycle. Other limitations include a relatively small sample size and the lack of a COWhen the participants ingested the thermogenic dietary supplement treatment, they experienced greater elevations in RMR values as compared to baseline, whereas no elevations in RMR were observed following the placebo treatment. These elevations came with no adverse effects relative to resting heart rate and diastolic blood pressure values, but due to the elevations in systolic blood pressure, caution should be taken for those with increased risk for hypertension or pre-hypertension. Taken on a daily basis, FitMiss Burn™ supplementation may increase overall energy expenditure, possibly leading to reductions in fat mass over time. Future work should investigate the effectiveness and safety of ingesting the dietary supplement over a longer period of time  to determine if reductions in fat mass are observed.


Noise-induced hearing loss is a common type of hearing loss. The effects of laser therapy have been investigated from various perspectives, including in wound healing, inflammation reduction, and nerve regeneration, as well as in hearing research. A promising feature of the laser is its capability to penetrate soft tissue; depending on the wavelength, laser energy can penetrate into the deepest part of the body without damaging non-target soft tissues. Based on this idea, we developed bilateral transtympanic laser therapy, which uses simultaneous laser irradiation in both ears, and evaluated the effects of bilateral laser therapy on cochlear damage caused by noise overexposure. Thus, the purpose of this research was to assess the benefits of simultaneous bilateral laser therapy compared with unilateral laser therapy and a control. Eighteen Sprague-Dawley rats were exposed to narrow-band noise at 115 dB SPL for 6 h. Multiple auditory brainstem responses were measured after each laser irradiation, and cochlear hair cells were counted after the 15th such irradiation. The penetration depth of the 808 nm laser was also measured after sacrifice. Approximately 5% of the laser energy reached the contralateral cochlea. Both bilateral and unilateral laser therapy decreased the hearing threshold after noise overstimulation in the rat model. The bilateral laser therapy group showed faster functional recovery at all tested frequencies compared with the unilateral laser therapy group. However, there was no difference in the endpoint ABR results or final hair cell survival, which was analyzed histologically. The type of threshold shift is determined by the intensity and duration of exposure. Several studies with similar levels of noise and exposure times  have reported that a PTS occurred after a few minutes or hours of such noise exposure , 2006. BLaser therapy has been used as a treatment for various symptoms, and its use has been increasing because of its non-invasive nature. After it was approved by the United States Food and Drug Administration, applications of laser therapy have widened in research scope, including wound healing , 1981, iOne useful feature of the laser is the capability to penetrate soft tissue; depending on the wavelength, laser energy can penetrate into deep parts of the body without damaging non-targeted soft tissues. This enables the delivery of laser energy from multiple points, which may lead to faster or increased effects of the laser in the target area. In our previous animal experiments, we found improvements in the hearing threshold not only in the laser-irradiated group but also in the contralateral ear , 2012. Tn = 6), unilateral laser (n = 6), and bilateral laser (n = 6)). All animals were treated in accordance with the Guide for Care and Use of Laboratory Animals , as formulated by the Institute of Laboratory Animal Resources of the Commission on Life Sciences. All procedures were approved by the Institutional Animal Care and Use Committee for Dankook University (DKU-15-048).Male Sprague Dawley (SD) rats (180–200 g) were used in this study. Eighteen rats were randomly divided into three different groups (noise only (The acoustic stimulus was a narrow band of noise which has frequency information centered at 16 kHz with 1 kHz of bandwidth (116 dB SPL). Rats were placed in individual cages to prevent defensive behaviors and these cages were placed in acryl reverberant chambers with a speaker BEYMA CP800Ti  attached on top. The traumatic stimulus was generated with a type 1027 sine random generator  and amplified with a R300 plus amplifier  for 6 h. For real time monitoring, a frequency-specific sound level meter  was used to monitor noise level in the chamber (placed on the floor) every hour so that consistent intensity (116 dB SPL) was maintained during noise exposure.Auditory brainstem responses (ABR) were measured to identify degrees of hearing loss and recovery. The evoked response signal-processing system  was used for ABR measurement. Animals were anesthetized with Zolazepam  and Xylazine  and placed in a sound proof chamber. Three needle electrodes were inserted at vertex (active) and beneath of each pinna (reference and ground), subcutaneously. The tone-burst stimuli  were used for experimental measurements and a total 1,024 responses were averaged. Responses were measured in 5 dB intervals from 90 to 10 dB SPL and thresholds were determined by the presence of peak within each signal. Hearing thresholds were obtained before and after noise exposure . ABR mea2, 594 J) for 15 days. The density of the laser was calibrated with a laser power meter  and detector sensor . The optical fiber  was attached to a hollow tube and placed into the external ear canal while leaving a distance of 1 mm between the fiber tip and tympanic membrane. Laser irradiation was performed on both the right and left ear simultaneously for the bilateral group and only in the right ear for the unilateral group. The noise only group was anesthetized and the optical fiber was placed into the external ear canal without power. Additional detailed information of the laser is described in An 808 nm diode laser  was used for laser therapy. Each rat in the experimental group was anesthetized and irradiated for 60 mins  were removed to expose the cochlea. The exposed contralateral cochlea was placed above the laser detector and the laser was used to irradiate the ipsilateral external canal with the protocol explained above.Laser energy was first measured from the contralateral side of the ear using an 808 nm laser irradiation  in the SD Rat to confirm the delivery of laser energy to the contralateral cochlea. The rat was sacrificed in a COFor the quantitative analysis of outer hair cells (OHCs), whole mounts of the organ of Corti were prepared. Intracardiac perfusion was performed using 4% Paraformaldehyde (PFA) followed by 0.9% normal saline. The cochlea was then harvested. After harvesting, the cochlea was fixed in 4% PFA overnight. After washing with 0.1 M Phosphate-buffered saline (PBS), the cochlea was decalcified with ethylenediaminetetracetic acid  and was dissected into three parts. The samples were prepared by staining with Phalloidin  and rinsed with 1x PBS. The samples were carefully examined under confocal microscopy  at a magnification of 400X.http://rsb.info.nih.gov/ij/) was used to count the number of cells in each section.We chose three representative areas for the quantitative analysis of OHC, which were located at 20, 50, and 80% from the apex, representing 4, 12, and 32 kHz respectively . Hair ceAll data were analyzed statistically using the Statistical Package for the Social Sciences software . We performed a Tuckey post hoc test following a Two-way Analysis of Variance (ANOVA) to determine the significance between hearing threshold for ABR measurement and number of hair cells.The 808 nm laser energy was first measured in the contralateral ear. Using an open air setup between the laser probe and detector, the energy output was determined to be the same at the detector and the displayed output of the laser. A total of 6 mW of laser energy was measured in the detector at the contralateral ear, while the maximum level of laser energy penetrating the contralateral ear was found to be 8 mW. This result suggests that some laser energy was absorbed prior to exiting the other ear .ABRs were measured before noise exposure to determine the baseline hearing threshold. Mean values (SDs) were 18.61 (5.37), 16.11 (5.57), 16.94 (6.67), 16.11 (5.3), and 16.39 (6.14) at frequencies of 4, 8, 12, 16, and 32 kHz, respectively . At 24 hp = 0.001 at 16 kHz and 0.046 at 32 kHz; p = 0.009 at 4 kHz, 0.04 at 8 kHz, <0.001 at 12 kHz, 0.001 at 16 kHz, and <0.001 at 32 kHz), and the difference between the unilateral group and noise-only group increased to 4 kHz . The rest 32 kHz) after tht 32 kHz) and 2D. to 4 kHz . This reA significant difference in the threshold between the bilateral group and the noise-only group was observed from the point of the sixth laser irradiation (at 16 kHz and 32 kHz)  and 2E. p = 0.0052 and 0.0006, respectively; A confocal image of three representative areas is presented in Cochlear damage can be variable, and a hearing threshold shift can occur abruptly or progressively, depending on the intensity and duration of noise overstimulation , 1991. IApplication of laser therapy, after noise overstimulation, induced recovery of hearing function similar to our previous study , 2012. TDespite the low penetration level in the contralateral ear, we found faster hearing recovery in the bilateral laser therapy group compared to the unilateral laser therapy group. Additional laser energy may improve the speed of hearing recovery by prompting the endo-organs of the contralateral cochlea. We hypothesize that the laser energy penetrating directly can affect the contralateral cochlea as an activator of cell metabolism. Additionally, the amount of laser energy in the middle of the head, which would be more than that reaching the contralateral cochlea, could have sufficient influence to activate the cochlear nerve or auditory pathway in the midbrain. Moreover, protective actions of cochlear efferent feedback pathway through olivocochlear bundle may also increase recovery. Several studies suggest that the protective effect of the olivocochlear bundle during hearing is allowed to suppress hyper activations of auditory nerve fibers and IHCs , 1988. AThe faster effect of bilateral laser therapy versus unilateral laser therapy is promising for clinical use. Most treatments after hearing loss due to different insults, require early intervention , 1960. TTo apply bilateral laser therapy in the clinic, some practical issues must be considered. Because of anatomical differences between humans and rodents, the effects of laser energy on the contralateral side would be different in humans compared to rodents. The larger distance from one ear to the other may limit the delivery of laser energy; however, the beneficial effect of bilateral laser therapy would be expected to remain if the mechanism involves targeting the brainstem. Increasing the power of the laser may be another approach to deliver energy to the other ear, but this could cause unwanted side effects, resulting in local burning and tympanic perforation. Thus, increasing the power of transcanal laser irradiation should be carefully considered before translation to clinical application.The present study showed positive effects of bilateral laser therapy after noise-induced hearing loss in an animal model. The results suggest that the use of bilateral laser therapy in a clinical setting may improve the therapeutic effects on hearing while minimizing side effects.10.7717/peerj.2252/supp-1Data S1Click here for additional data file.10.7717/peerj.2252/supp-2Data S2Click here for additional data file.