Triclosan (TCS), an antibacterial agent, is identified in serum and urine of humans. Here, we show that the level of urinary TCS in 28.3% patients who had spontaneous abortion in mid-gestation were increased by 11.3-fold (high-TCS) compared with normal pregnancies. Oral administration of TCS (10 mg/kg/day) in mice (TCS mice) caused an equivalent urinary TCS level as those in the high-TCS abortion patients. The TCS-exposure from gestation day (GD) 5.5 caused dose-dependently fetal death during GD12.5–16.5 with decline of live fetal weight. GD15.5 TCS mice appeared placental thrombus and tissue necrosis with enhancement of platelet aggregation. The levels of placenta and plasma estrogen sulfotransferase (EST) mRNA and protein in TCS mice or high-TCS abortion patients were not altered, but their EST activities were significantly reduced compared to controls. Although the levels of serum estrogen (E2) in TCS mice and high-TCS abortion patients had no difference from controls, their ratio of sulfo-conjugated E2 and unconjugated E2 was reduced. The estrogen receptor antagonist ICI-182,780 prevented the enhanced platelet aggregation and placental thrombosis and attenuated the fetal death in TCS mice. The findings indicate that TCS-exposure might cause spontaneous abortion probably through inhibition of EST activity to produce placental thrombosis. Spontaneous abortion is the most common complication of human pregnancy and 10–15% of clinical pregnancies end in it35et al. has reported that TCS can inhibit the activity of sheep placental cytosolic EST to reduce the levels of both estradiol and estrone sulfonationSULT1E1, is expressed in human, bovine and murine placentas during mid- to late gestation to catalyze the sulfoconjugation of estrogen (E2) at the 3-hydroxyl position1314There are several biological activities of TCS that are unrelated to its antibacterial action. For example, TCS reduces the level of thyroid hormone in weanling rats8To determine the influence of TCS on the process of pregnancy, an epidemiological investigation was primarily designed to examine the level of urinary TCS in normal pregnancy and spontaneous abortion patients in mid-gestation. According to the level of urinary TCS in spontaneous abortion patients, we prepared the model of pregnant mouse exposed to TCS, in which the fetal survival and development were examined during mid- to late gestation. In addition, placental structure, the levels of reproductive hormones and thyroid hormones, the expression and activity of EST were further examined in spontaneous abortion patients and TCS-exposed pregnant mice. Our results indicated that the exposure of TCS in humans and mice might cause spontaneous abortion in mid-gestation probably through the inhibition of EST activity leading to placental thrombosis and degeneration.et al.P = 0.492). Meanwhile, there was a difference in the body mass index (BMI) between abortion patients (21.89 ± 2.60 kg/m2) and controls . In abortion patients, the detectable rate of urinary TCS  was increased approximately 11.3-fold compared to mean of controls (0.99 ng/ml).Measuring TCS and its metabolites in urine represents an important biomonitoring tool for exposure assessment 57.52%;  was 1.762 = 1, n = 20). However, the level of urinary TCS showed no significant difference between GD10.5 and GD15.5 10-TCS mice (data not shown).To examine the influence of TCS on the maintenance of pregnancy and fetal development, 3-month-old mice were treated with TCS daily by gavage at doses of 1, 10 and 100 mg/kg/day  from gestation day (GD) 5.5. The level of urinary TCS was measured at GD10.5 and GD15.5, respectively. As shown in P < 0.001, n = 20; P = 0.030, n = 20), respectively. The incidence of spontaneous abortion in 100-TCS mice and 10-TCS mice reached 80% , respectively. In addition, the body weight of live fetuses was significantly reduced in 100-TCS mice  compared with controls.The TCS-exposure caused a dose-dependent increase in the fetal death . In compP = 0.015, n = 10) and was gradually aggravated during GD14.5  and GD16.5 . Consistently, the number of dead or reabsorbed fetuses showed a progressive increase from GD13.5  to GD16.5 .To determine the time of TCS-induced spontaneous abortion, we further examined the numbers of live and dead fetuses during GD10.5-16.5. As shown in P = 0.015, n = 20; Notably, the placentas were dark in GD15.5 10-TCS mice and 100-TCS mice, but not in 1-TCS mice . The plaP = 0.358, n = 20; P = 0.196, n = 20) and β-HCG  in GD12.5 TCS mice did not differ significantly from control mice. The levels of P4  and β-HCG  were significantly reduced in GD15.5 100-TCS mice compared to controls, while E2 level had no significant difference between the both groups . However, the levels of sulfo-conjugated (S-E2) were reduced in TCS mice , while the levels of unconjugated E2 (free E2) were observably increased in TCS mice  compared to controls. Thus, the ratio of S-E2 and free E2 in TCS mice  was lower than that in control mice . In addition, the levels of serum thyroxine (T4) and triiodothyronine (T3) in TCS mice  were lower than those in control mice.The levels of serum estrogen (E2), progesterone (P4) and β-human chorionic gonadotropin (β-HCG) were investigated in 100-TCS mice and abortion patients. The levels of E2  and β-HCG  were clearly lower than those in normal gestation . The level of E2 in low-TCS abortion patients was lower than control , whereas in high-TCS abortion patients had no difference from control group .In the low-TCS and high-TCS abortion patients, the levels of P4 , GD12.5  and GD13.5  followed by a detectable decline at GD14.5-16.5 . Consistently, the levels of EST protein in placenta , although had no significant difference between GD12.5 100-TCS mice and control mice . The activity of EST was further examined by radiochemical extraction assay when the amount of EST protein was corrected to be equal. Notably, the activities of placental and plasma EST in either GD12.5  were significantly reduced compared to control mice.The level of placental t GD15.5 . In compplacenta  or plasmplacenta  in GD15.P = 0.013, n = 10; P < 0.001, n = 10). Although the EST protein in high-TCS abortion patients tended to be less than that in low-TCS abortion patients, the group comparison failed to reach significance . Importantly, the activity of plasma EST in high-TCS abortion patients was lower than in control group .In comparison with control group of normal gestation 22–24 weeks, the level of plasma EST protein was reduced in low-TCS  were increased in 100-TCS mice with the decline of prothrombin time (PT)  and activated partial thromboplastin time (APTT)  compared to controls, which could be corrected by the estrogen receptor (ER) antagonist ICI 182,780 when administered on GD12.5-15.5 . As expected, the administration of ICI 182,780 prevented the placental thrombosis in GD15.5 10-TCS and 100-TCS mice  (TCS mice  and partin vivo evidence that exposure to TCS can cause spontaneous abortion in humans and mice. This conclusion is deduced mainly from the following results. The level of urinary TCS in 28.3% high-TCS spontaneous abortion patients (11.21 ng/ml) was increased by 11.3-fold compared to normal pregnancies (0.99 ng/ml). The National Health and Nutrition Examination Survey collected 2,517 urine samples in a representative US population and detected urinary TCS in 74.6% of the samples at concentrations of 2.4–3790 μg/lThe present study provides the first SULT1E1 expression has been determined in the placenta of humans, cows and mice during mid- to late gestation1314et al.SULT1E1 mRNA and EST protein in GD12.5 did not differ significantly from control mice, while in GD15.5 were reduced compared to controls. One possibility is that the placental tissue necrosis in GD15.5 100-TCS mice leads to the reduction of EST. In addition, the level of plasma EST protein had no significant difference between low-TCS and high-TCS abortion patients, although it was lower than that in control group. It is conceivable that the TCS-exposure does not affect the EST expression in mice and human. One principal finding in this study is that the activity of EST was significantly reduced in high-TCS abortion patients and GD12.5-15.5 TCS mice. James et al. have reported that the inhibitory effect of TCS on sheep placental EST activity was mixed competitive/uncompetitive actionsSULT1E1 knockout mice from DG12.5 to DG 17.5 were significantly elevated, which were associated with the increase in the levels of free E2 in the amniotic fluids at DG14.5 and DG17.5Placenta provides nearly all of E2 in the forms of sulfo-conjugated E2 (S-E2) and estrone, which are present at much higher concentrations than the unconjugated E2 (free E2). The discrete localization of EST at the interface of the fetal-maternal blood exchange suggests that EST may play a critical role in modulating E2 activity in both fetal and maternal plasma. The levels of serum E2 in SULT1E1 knockout mice showed placental thrombus and fetal death during mid- to late gestation16Another important finding in the present study is that the platelet aggregation and platelet activation were enhanced in TCS mice, which could be blocked by the ER antagonist. In particular, the blockade of ER reduced remarkably the placental thrombus and hemorrhage in GD15.5 TCS mice. The over-activation of ER has been reported to induce platelet aggregation in human through cascading Src, Pyk2 and PI3-kinase or increasing the integrin affinity for fibrinogen to reduce the threshold of platelet activation2324et al. have reported the presence of estradiol-3-sulfate in the plasma of fetal sheep at concentrations of approximately 1 ng/ml28Interestingly, the ER antagonist could attenuate the fetal death in TCS mice. A possible explanation is that the increase of activated E2 in TCS mice causes the placental degeneration and failure leading to spontaneous abortion. The delivery of oxygen and nutrients to the developing fetus is highly dependent on the maintenance of high uterine blood flow. Wood et al. reported that the concentration of TCS that causes substantial inhibition of EST is in the range of the environmental exposure levelin vivo evidence that the exposure of human or mice to TCS, at least partially, through inhibiting EST activity might cause spontaneous abortion and growth retardation of fetuses. This study is the first to implicate that the activation of EST is required for the maintenance of pregnancy and fetal development during mid to late gestation in human.TCS is widely used in personal care products. Stanley The subjects were volunteers from the NJMU Birth Cohort at affiliated hospitals of Nanjing Medical University. The Institutional Review Board of Nanjing Medical University approved the protocols. All of the studies involving human subjects were performed under full compliance with government policies and the Helsinki Declaration. After explanation of the study procedures and clarification of the questions raised, all of the subjects gave their informed consent. A complete physical examination, including measurement of the height and weight, was performed. A questionnaire was used to collect information, including personal background, lifestyle factors, occupational and environmental exposures, genetic risk factors, sexual and reproduction statuses, medical history, and physical activity. Controls were females who had no history of spontaneous abortion and had at least one living child. The cases were females who had medically unexplained spontaneous abortion in mid-gestation weeks 14–24The urinary concentration of TCS was measured with ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) . Firstly, urine samples were incubated in 1 M ammonium acetate buffer solution (pH = 5.0) for hydrolyzation with β-glucuronidase/sulfatase (20000 units/ml) overnight. After hydrolysis, the phenols were extracted and preconcentrated with solid phase extraction (SPE) , and determined with UPLC  electrospray ionization (negative ion mode)-MS/MS. The limit of detection (LOD) was 0.9 ng/ml. The intra- and inter-day precisions for TCS were between 9% and 38% and the recoveries were between 99% and 138% at spiked concentration of 2, 20 ng/mL. Quality control samples were analyzed in parallel with unknown samples in each analytical series. Creatinine (CR) concentrations were analyzed for correcting the phenols concentration variations caused by fluctuated urine concentration and dilution. CR concentrations of urine were measured with an automated chemistry analyzer .All of the animal experimental procedures followed the guidelines of the Laboratory Animal Research of Nanjing Medical University and were approved by the Institutional Animal Care and Use Committee of Nanjing Medical University. Three-month-old female and male mice , weighing approximately 30–35 g, were used in the study. All of the mice were housed under a 12/12 hour light/dark cycle (lights on at 0600 hour) with free access to food and tap water. Detection of a vaginal plug was chosen to indicate day 0.5 of gestation (GD0.5). TCS  was dissolved in dimethylsulfoxide (DMSO), and then diluted was with corn oil. The dams were given oral administration of TCS at doses of 1, 10 and 100 mg/kg per day. Control mice were treated with vehicle at the same volume. During the treatment with TCS, the dams did not show significant abnormality in body weight. A pellet of the ER antagonist ICI182,780  with 1.5 mg/12 days of releaseThe placenta was fixed in 4% paraformaldehyde and then dehydrated through a graded series of alcohol, cleared in xylene and embedded in paraffin wax. Sections (5-μm thick) deparaffinized were stained with hematoxylin and eosin (HE) and observed using a conventional light microscope .et al.The blood samples were collected within 12 hours after spontaneous abortion in humans. Orbital blood (500 μl) of mice was obtained under anesthetized conditions with chloralhydrate  at 9 hours 1600–1700) after the administration of TCS. Levels of serum total E2, progesterone (P4), β-human chorionic gonadotropin (β-HCG), thyroxine (T4) and triiodothyronine (T3) were examined using a radioimmunoassay (RIA) kit provided by the National Hormone and Peptide Programme . The intra- and inter-assay coefficients of variation were 6% and 5.8% for E2, 5.8% and 8.4% for P4, 3.4% and 12.3% for β-HCG, 5.1% and 8.6% for T4, 2.9% and 8.3% for T3, respectively. The lowest detectable levels were 2 pg/ml for E2, 0.2 ng/ml for P4, 2 mIU/ml for β-HCG, 5.0 ng/ml for T4, 0.1 ng/ml for T3, respectively. The plasma was extracted with ethanol to preserve the water-soluble steroid including sulfo-conjugated E2 (S-E2) or using hexane/ethyl acetate  to obtain unconjugated E2 (free E2), according to the method of Wood 00–1700 aet al.The anticoagulated blood samples using sodium citrate were collected. Fibrin degradation product (FDP) is measured by Cusabio rat FDP ELISA assay kit  according to the manufacturer’s instructions. To estimate platelet aggregation, platelet rich plasma (PRP) was prepared by centrifugation (250 g) for 10 min, and then tested by the turbidimetric technique according to the method of Mustard We performed Western blot analysis as described previouslySULT1E1 and GAPDH mRNA were designed according to earlier publicationsTotal RNA was extracted from the placentas using Trizol  according to the manufacturer’s instructions. RNA (1 μg) was used for reverse transcription using high-capacity cDNA of the reverse transcription kit RT  according to the instructions. The primer sequences of 3H-estradiol (60 Ci/mmol) was purchased from PerkinElmer Life Sciences Inc. . Incubation mixtures (0.5 ml) contained 100 mM Tris–HCl (pH 7.4), 5 mM MgCl2, 0.8 mg placental cytosolic protein, 20 μM PAPS, and 2 nM [3H] E2. The reaction was initiated with the addition of PAPS and continued for 30 minutes at 37 °C. The reaction mixture was extracted with 1 ml of dichloromethane and aliquot of the aqueous phase was counted by liquid scintillation counting.EST activities were measured using a radiochemical extraction assaypost-hoc analysis, or Student’s t test when appropriate. In some cases, logistic regression and chi-squared test were used. Differences at P < 0.05 were considered statistically significant.The group data were expressed as the means ± standard error (SE). All of the statistical analyses were performed using the SPSS 16.0 software . Differences among the means were analyzed using ANOVA, followed by Bonferroni How to cite this article: Wang, X. et al. Triclosan causes spontaneous abortion accompanied by decline of estrogen sulfotransferase activity in humans and mice. Sci. Rep.5, 18252; doi: 10.1038/srep18252 (2015).


Photoreceptors are damaged in many common eye diseases, such as macular degeneration, retinal detachment, and retinitis pigmentosa. The development of methods to promote the repair or replacement of affected photoreceptors is a major goal of vision research. In this context, it would be useful to know whether photoreceptors are capable of undergoing some degree of spontaneous regeneration after injury. We report a subject who lost retinal function in a wide zone around the optic disc, giving rise to massive enlargement of the physiological blind spot. Imaging with an adaptive optics scanning laser ophthalmoscope (AOSLO) showed depletion of cone outer segments in the affected retina. A year later visual function had improved, with shrinkage of the enlarged blind spot. AOSLO imaging showed repopulation of cone outer segments, although their density remained below normal. There was a one-to-one match between sites of formerly missing outer segments and new outer segments that had appeared over the course of the year’s recovery. This correspondence provided direct morphological evidence that damaged cones are capable, under some circumstances, of generating new outer segments. Rods and cones are specialized neurons, containing stacks of membrane laded with photopigment in a compartment called the outer segment, which is anchored by a cilium to the rest of the cell. They initiate the process of vision by converting patterns of light energy into graded electrical potentials. Diseases that affect photoreceptors are a major worldwide cause of vision loss. In 1988, a new syndrome called acute idiopathic blind spot enlargement (AIBSE) was described346789In these rare photoreceptor diseases, there is usually a substantial degree of vision recovery. The cellular events underlying this gradual improvement are obscure. One possibility is that some lost or damaged outer segments are replaced following photoreceptor damage.This study was conducted to determine whether, after injury, photoreceptors have the capacity for spontaneous regeneration of new outer segments. To address this issue we performed serial imaging using an adaptive optics scanning laser ophthalmoscope (AOSLO) in a human subject with a dense but transient visual field defect from AIBSE. Adaptive optics, by compensating for optical imperfections, allows one to resolve individual cones in the intact eye11An 18-year-old woman experienced flashing lights in the temporal field of her left eye for several weeks, along with onset of a dense scotoma. Although her acuity was normal, visual field testing  revealedWith a conventional ophthalmoscope, no abnormality could be detected in the appearance of the optic disc or retina in the left eye. Faced with compelling evidence of outer retinal dysfunction, AOSLO was performed to investigate the morphology of individual photoreceptors in the vicinity of the optic disc. Images were acquired along a 21° horizontal swath, which began temporally in the retina (8°), passed through the fovea (0°), and ended nasally at the optic disc (12.75°) . For com2. This finding showed that cone damage in AIBSE can be widespread, occurring at sites located far from the optic disc. Although nearly half the outer segments in this portion of the temporal retina appeared to be absent, there was only a 3–6 decibel (dB) loss of retinal sensitivity in this region on visual field testing  . There adiameter . This indiameter . InteresIn the AOSLO image acquired in 2014, half the missing outer segments were still missing . HoweverAs a control, it is useful to compare the stability of cone locations in normal retina over the course of a year. Cone profiles were plotted in the right eye , in a reLoss of cones has been shown with AOSLO in numerous retinal diseasesThe problem is compounded by the fact that individual cones undergo changes in reflectance over time, and therefore, for any given cone, one cannot assume that absence of light back scatter means that the outer segment is missingRecent evidence suggests that the reflectance of individual cones oscillates in a cyclical fashion, perhaps in relationship to disc sheddingThe most likely interpretation of our findings is that outer segments were destroyed in our subject, resulting in the dark gaps in the initial AOSLO images. This inference is supported by optical coherence tomography , which sAfter regeneration, the left retina contained about 50% of the normal concentration of outer segments at 6.5°, yet sensitivity to light was nearly normal, being reduced by only 3 dB. Relative sparing of light sensitivity, despite cone loss, has been reported previously in color blindness. For example, in deuteranopia a 30% loss of cones produces no visual field depression, unless gaps in the cone mosaic are probed with extremely small test spots22The retina is susceptible to mechanical injury, from blunt trauma (commotio retinae), subretinal hemorrhage, or retinal detachment. With re-apposition of the retina to the pigment epithelium and the passage of time, there is usually slow improvement in visual function. Regeneration of outer segments may contribute to this recovery, but would be difficult to show using AOSLO, because of the difficulty of obtaining satisfactory baseline images of a retina that has been disrupted physically. In a disease such as AIBSE, the situation is auspicious for AOSLO imaging, because vision loss does not follow a mechanical insult to the retina.In an animal model of hereditary retinal disease, ciliary neurotrophic factor has been shown to reverse the degeneration of outer segments25All research procedures were approved by the Committee on Human Research at the University of California San Francisco. The subject provided informed written consent for participation. All examinations and tests were carried out in accordance with the approved guidelines. The subject was an 18-year-old woman in excellent health who developed a spontaneous temporal visual field defect in her left eye from a syndrome known as acute idiopathic blind spot enlargement. She had no other history of illness, hospitalization, or treatment with any medication. Her visual acuity was 20/15 in each eye and there was no refractive error. An afferent pupil defect was present in the left eye. Slit lamp examination and fundus examination with a direct ophthalmoscope were normal. A fluorescein angiogram was also normal (not illustrated). After her initial examination, the patient was seen periodically over the next year to monitor her recovery.Automated perimetry was performed with a Humphrey Field Analyzer  using a full threshold algorithm and a size III (0.43°) white stimulus. No refractive correction was necessary. On each occasion, the central 30° and the peripheral 30–60° of the visual field were tested in each eye.A Heidelberg Spectralis  instrument was used to obtain spectral domain optical coherence tomographic (OCT) images of each retina, both at the time of her initial evaluation and each follow-up visit.2, with light tiles measuring 200 cd/m2 and dark tiles 4 cd/m2. An infrared camera was used to monitor fixation. Responses were recorded during 16 sequences, each lasting 30 sec. The signal was amplified by 105, band-pass filtered between 10–100 Hz, and spatially averaged at 17%.A multifocal electroretinogram (mfERG) was obtained using a VERIS Clinic 5.1.10X model instrument , following standards of the International Society for the Clinical Electrophysiology of Visionwww.virtualdub.org). The remaining frames were averaged to maximize the signal-to-noise ratio . The final image was assembled into a montage by translation and rotation to match overlapping features in adjacent images. The resulting montage was then positioned on a photograph of the retina by using blood vessels for alignment .To probe the cause of visual field loss in acute idiopathic blind spot enlargement, AOSLO was used to acquire images of the photoreceptor mosaic after pharmacological dilation of the pupils. Both imaging sessions were conducted in the early afternoon. The subject’s head was stabilized in a chin/forehead rest while she fixated a green 0.20° laser target spot. The imaging system consisted of a low-coherence, 840-nm light source, a Shack-Hartman wavefront sensor, and a 140-actuator microelectromechanical deformable mirror. A 10 sec, 300 frame, 512 × 512 pixel video subtending approximately 1.2 × 1.2° was recorded at each location, beginning in the fovea. Subsequent videos were recorded by displacing the fixation target along the horizontal meridian of the visual field in 0.5–1.0° steps. Image offset and warp, which occur from frame to frame due to fixation instability, were corrected with custom software employing a map-seeking circuit algorithmTo measure cone density, a grid was superimposed on each montage to divide it into tiles measuring 100 × 100 pixels (420 pixels/deg). Tiles that contained a portion of a blood vessel which obscured the cone mosaic were eliminated. In addition, tiles that extended beyond the edge of the montage were eliminated. The number of cones in each of the remaining tiles was counted manually. The identification of cone outer segments, and the empty silos left by missing outer segments, depended on observer judgement. All the AOSLO images used to compile the measurements of cone density in A total of 773 tiles was analyzed and 36,141 cones were counted. The density of cones was calculated for each tile. The result was plotted for each montage as a function of distance from the fovea  The distThe eyes from a 37-year-old woman with normal visual function who died from idiopathic pulmonary fibrosis were removed 6 hours after death and placed into 4% paraformaldehyde. Each retina was dissected from the pigment epithelium and flat-mounted onto a glass slide, with the photoreceptor layer facing up. The retina was cleared with dimethyl sulfoxide and coverslipped with glycerin. Stacks of images of photoreceptor processes were obtained at different eccentricities between the fovea and optic disc in each eye using an inverted research microscope (Nikon Eclipse Ti) equipped with Nomarski interference contrast optics. Each image had a depth of field of 1.44 μm and individual images were obtained every 0.5 μm in the Z-plane. Imaging from the tips of the photoreceptors to the external limiting membrane yielded about 70 images, about half passing through outer segments and half through inner segments.How to cite this article: Horton, J. C. et al. Spontaneous Regeneration of Human Photoreceptor Outer Segments. Sci. Rep.5, 12364; doi: 10.1038/srep12364 (2015).


Hearing plays a vital role in the performance of a soldier and is important for speech processing. Noise-induced hearing loss is a significant impairment in the military and can affect combat performance. Military personnel are constantly exposed to high levels of noise and it is not surprising that noise induced hearing loss and tinnitus remain the second most prevalent service-connected disabilities. Much of the noise experienced by military personnel exceeds that of maximum protection achievable with double hearing protection. Unfortunately, unlike civilian personnel, military personnel have little option but to remain in noisy environments in order to complete specific tasks and missions. Use of hearing protection devices and follow-up audiological tests have become the mainstay of prevention of noise-induced hearing loss. This review focuses on sources of noise within the military, pathophysiology and management of patients with noise induced hearing loss. Noise-induced hearing loss is a major preventable disease. It can be caused by an acute exposure to an intense impulse of sound or by a continuous steady-state long-term exposure with sound pressure levels higher than 75–85 dB Table .Table 1Noise remains a large public health problem with an estimated 1.3 billion people being affected by hearing loss [The prevalence of hearing loss and tinnitus in military population are greater than in the general public. Almost every soldier, sailor, airman or marine will be exposed to hazardous noise levels at some point in their career -4. The tHearing acuity is a key component of a soldier’s effectiveness in the battlefield. The presence of tinnitus and hearing loss can significantly impair a soldier’s ability to hear important acoustic cues or communication signals from the unit or the enemy . HearingSources of noise within the military vary with soldier’s designation. Within the Belgian military, Fighting in Built-Up Area (FIBUA) training, shooting with large calibre weapons and participation in military exercises were the strongest determinants of hearing loss .Within the infantry, weapons emit high levels of noise. Table In the Navy, the highest indoor noise levels were found in engine rooms ,10. LandMilitary aircraft personnel are not spared, the average noise experienced in service helicopters was found to be 97 dBA for ‘Gazelle’ , 99.8 dBA for the ‘Scout’ , 99.9 dBA for the ‘Puma’ and 100 dBA for the ‘Lynx’ . In fighInjury from noise can occur in 2 main ways. First, high level, short duration exposure exceeding more than 140 dB can cause the delicate inner ear tissues to beyond stretch beyond their elastic limits. This causes mechanical disruption of the sterocilia and direct damage to supporting and sensory cells . This tySecond, long term exposure to low level noise damages the cochlea metabolically rather than mechanically. It involves biochemical pathways leading to cell death either through apoptosis or necrosis . There aExposure to intense sound can cause auditory thresholds to become elevated permanently or temporarily. Reversible hearing loss is referred to as temporary threshold shift (TTS). Depending on duration of exposure, recovery from TTS can occur over a period of minutes to hours or days. If TTS does not recover, permanent hearing loss results and this is referred to permanent threshold shift (PTS) . These tPTSs are postulated to be either due to direct mechanical trauma or metabolic overstimulation of cellular elements within the organ of Corti which is associated with generation of reactive oxygen species .Various mechanisms have been proposed for TTS and include synaptic fatigue, metabolic fatigue of either stria vascularis or hair cells and changes in cochlear blood flow. An important component of noise-induced hearing loss is postsynaptic damage in the afferent dendrites beneath the inner hair cells . Even thNoise not only increases hearing threshold, but it can also cause tinnitus and hyperacusis. This can be present in individuals with normal hearing thresholds but with cochlear neuropathy. Indeed, studies have shown that patients with tinnitus have evidence of reduced Wave I at high sound levels ,27. The Exposure to noise can induce several hearing symptoms such as temporary threshold shifts (TTS), tinnitus, hyperacusis, recruitment, distortion or abnormal pitch perception . TinnituPatients may exhibit difficulty in listening to high frequency noise such as whistles or buzzers. They may also have difficulty differentiating some speech consonants, especially if they are in areas where there is significant background noise.However these symptoms are typically insidious and most patients with noise induced hearing loss may not notice their deficiency until it starts to affect communication.Noise-induced deafness usually occurs at high frequencies with hearing loss beginning around 4 kHz or 6 kHz. However, as the disease progresses, hearing loss will also be seen at the lower frequencies. The expected maximal changes in thresholds are predictable at one-half octave above maximal frequency of the exposure .The audiometric pattern in noise induced hearing loss is usually symmetrical and bilateral. However some asymmetry is not unexpected. The asymmetry in hearing threshold may be partly explained by the position of head during work . Hong etWithin the military setting, noise exposure may be controlled through isolation , vibration dampening, insulation and proper equipment maintenance . The preCurrently, the Navy considers 85dBA to be the threshold for single hearing protection and 104 dBA for double hearing protection for steady state noise settings . Noise let al. found that hearing was better than predicted in nearly all frequencies for both ears for both Lynx and Apache pilots, demonstrating that the circumaural earmuffs implemented reduce the risk of noise induced hearing loss [In the British Army Air Corps, pilots of the Lynx have to wear the Mk4 flying helmet and pilots of the Apache wear the Integrated Helmet and Display Sighting System (IHADSS). Circumaural earmuffs are integrated into the aircrew helmet system. Lang ing loss .et al. conducted a study on the hearing protection equipment use by the crew on the flight deck and found that 79% of flight deck personnel received an estimated 0–6 dB rather than the expected 28–30 dB of noise attenuation from either misuse of earplugs or non-compliance to ear plugs [Even the best hearing protection equipment will be ineffective if it is not used properly or if soldiers are not compliant. A focus group study found that main concerns with hearing protection were interference with detection and localization of auditory warning and perception of orders . Bjorn ear plugs .Currently there is no established treatment for patients and it is limited to prevention and follow-up. However recent clinical trials have proved promising.Magnesium efficacy was tested in a double-blind study. Test subjects were given either 122 mg of magnesium or a placebo for 10 days and thereafter subjected monoaurally to 90 dB SPL of white noise for 10 minutes. TTS of > 20 dB was found in 28% of the placebo group compared to 12% in the magnesium-supplemented group .et al. conducted a double-blind placebo controlled study on army recruits and concluded that recruits who had magnesium supplementation had less frequent noise-induced PTS compared to the placebo group [Attias bo group . These 3NAC acts as a reactive oxygen species scavenger and is postulated to reduce noise-induced hearing loss by reducing the exposure of the cochlea to reactive oxygen species. Glutathione S-transferases (GST) are a family of detoxification enzymes which help cells resist oxidative injury. Glutathione detoxification can be affected in individuals with genetic polymorphisms involving deletion of base pairs in the genes like GSTT1 and GSTM1. Patients with these two high-risk genotypes are more prone to have oxidative injury from noise induced hearing loss ,45. In aAnother glutathione (GSH) precursor is MET, an essential amino acid that can be converted to cysteine, which is the rate-limiting substrate for GSH production. It has been shown in animal studies to be otoprotective when administered at 200 mg/kg . A majorEbselen is a potent glutathione peroxidase mimic and neuroprotectant. It also has strong activity against peroxynitrite, a super reactive oxygen species ,49. It rNoise-induced hearing loss is a serious disease burden in the military. Due to the nature of the military profession, hearing is a vital asset during tactical and survival training and exposure to loud noises during training and missions are inevitable. Prevention is still the mainstay of treatment and soldiers need to be educated with regards to the use of hearing protection devices.


Colletotrichum.Horizontal gene transfer (HGT) is the stable transmission of genetic material between organisms by means other than vertical inheritance. HGT has an important role in the evolution of prokaryotes but is relatively rare in eukaryotes. HGT has been shown to contribute to virulence in eukaryotic pathogens. We studied the importance of HGT in plant pathogenic fungi by identifying horizontally transferred genes in the genomes of three members of the genus Colletotrichum or their ancestors. The HGT events include genes involved in amino acid, lipid and sugar metabolism as well as lytic enzymes. Additionally, the putative minimal dates of transference were calculated using a time calibrated phylogenetic tree. This analysis reveals a constant flux of genes from bacteria to fungi throughout the evolution of subphylum Pezizomycotina.We identified eleven HGT events from bacteria into members of the genus Genes that are typically transferred by HGT are those that are constantly subject to gene duplication and gene loss. The functions of some of these genes suggest roles in niche adaptation and virulence. We found no evidence of a burst of HGT events coinciding with major geological events. In contrast, HGT appears to be a constant, albeit rare phenomenon in the Pezizomycotina, occurring at a steady rate during their evolution.The online version of this article (doi:10.1186/1471-2164-16-2) contains supplementary material, which is available to authorized users. Horizontal gene transfer  is the stable transmission of genetic material between organisms without the use of vertical inheritance mechanisms, mitosis or meiosis . HGT is The genetic mechanisms that are responsible for HGT are not well known. The nuclear envelope, the storage of DNA in chromatin, RNA interference systems, separate reproductive cell lines, gene promoter specificity, incompatibility of intron splicing systems, alternative gene codes and others represent barriers against HGT especially in distantly related species (e.g. inter-kingdom HGT) –16. The A wide range of methods have been proposed to detect HGT including phylogenetic analysis, and the detection of bias in nucleotide composition and codon usage, using naïve Bayes classifiers, correspondence analysis, or Akaike information criterion clustering . Each meColletotrichum, is one of the most destructive, causing significant crop losses worldwide [Colletotrichum fungi are important as experimental models in studies of many aspects of plant disease [Colletotrichum and are providing new insight into the study of plant-fungal interactions, , including the evolution of pathogenicity [Colletotrichum plant-like subtilisin) that was laterally transferred from plants to an ancestor of Colletotrichum[Colletotrichum to identify additional evidence of HGT to determine the impact of HGT on the evolution of pathogenicity in filamentous fungi. We discuss the potential role of the candidates in pathogenicity and niche adaptation. We found that genes typically transferred by HGT are those that belong to families that are subject to constant gene duplication and loss. We also determined the age of the HGT events, by means of a time calibrated phylogeny, and discuss the timing of HGT events within the context of major geological events. This is the first time that the impact of HGT has been evaluated on a genome-wide scale in Colletotrichum, and gives us insight into the evolution of this important genus.Among the plant diseases caused by fungi, anthracnose caused by members of the fungal genus orldwide –26. Coll disease –30. Drafgenicity . Previouotrichum. In the Colletotrichum species (C. graminicola M1.001 [C. higginsianum IMI 349063 [C. gloeosporioides Cg-14 [http://www.uniprot.org). Next, we selected proteins that having at least 80% of the top 120 hits  with a taxonomic classification other than fungi as candidates for further analysis. The threshold of 80% was selected by evaluating previously described HGT candidates reported by Richards et al. [The most robust method to detect HGT is phylogenetic analysis . Therefoa M1.001 , C. higgI 349063  and C. ges Cg-14 ), we devs et al.  and Richs et al.   using the contig sequences as queries. We found that the contigs align to the Bacillus pumilus genome over regions ranging from 203 to 1615 bp and sequence similarities ranging from 87.3% to 92.5% identity . Based on these BLAST searches, we concluded that these contigs are the result of bacterial contamination of the C. gloeosporioides genome assembly and we removed the corresponding HGT candidates from further analysis. We performed another set of BLAST searches to identify homologs to the HGT candidates in the three Colletotrichum genomes and then arranged these additional proteins and the HGT candidates into groups of homologous sequences. One group of homologous sequences is comprised of two subtilisin-like serine proteases putatively transferred from a plant ancestor and which we previously described as CPLSs (Colletotrichum Plant-Like Subtilisin) [During our analysis, we identified several proteins from tilisin) . The remtilisin)  and Marctilisin)  respectiC. graminicola HGT candidates of bacterial origin have introns consistent with their prokaryotic origin. Only 26.4% of the genes encoded in the C. graminicola genome lack introns and the probability of selecting 11 intronless genes at random from the C. graminicola genome is 4.06e-7 further supporting the hypothesis that the 11 candidates are of bacterial origin. To corroborate this calculate we performed a Mann–Whitney-Wilcoxon test to determine whether the two samples of genes (the whole genome and the HGT candidates) are the same, with respect to the number of introns in the genes. These two set of genes were significantly different (p ≈ 0), from which we conclude that the 11 HGT candidates have a different intron distribution than the genome.None of the 11 We considered that the HGT candidates might lack introns because they are members of gene families that typically lack introns. To test this alternative hypothesis, we first identified 75 homologs to the HGT candidates by searching the proteomes for proteins with the same functional annotation as the candidates. We counted the number of introns in the 75 homologs and compared this sample to the intron content of the rest of the genes in the genome using a Mann–Whitney-Wilcoxon test. The 75 gene sample was not significantly different from the rest of the genes in the genome (p = 0.33). Thus, we conclude that the HGT candidates are not from intron-poor gene families. This evidence supports the hypothesis that the lack of introns in the HGT candidates is the consequence of their bacterial origin.C. graminicola and the rest of the genes encoded in the genome. We found that the GC content of the HGT candidates is not different from the rest of the genome (p = 0.337).The GC content of horizontally transferred genes can be different than genes within the recipient genome . We usedC. graminicola genome share the same annotation.The putative function and the biochemical pathways of the HGT candidates were deduced with BRENDA Release 2012.02) , KEGG  is the only HGT candidate that does not have vertically transmitted homologs in the eactions , 43.Magnaporthe oryzae and other fungi [Cochliobolous heterostrophus, OXI1, is required for biosynthesis of the secondary metabolite T-toxin [Aspergillus fumigatus which plays a role in siderophore biosynthesis and subsequently, virulence [Fusarium oxysporum f. sp. melonis[F. oxysporum have reduced virulence, linking virulence with arginine biosynthesis.At least 4 HGT candidates have clear associations with plant interactions and virulence. Members of HGT4 encode secreted glycoside hydrolases belonging to CAZy family GH74  which deer fungi , 44, 45. T-toxin  and nullirulence . Finally melonis. Null muMany of the HGT candidates belong to functional categories that are described as enriched in ‘volatile’ genes by Wapinsky et al. . VolatilC. graminicola and C. higginsianum during three stages of infection of their respective hosts, maize and Arabidopsis. We also examined the expression of a selection of vertically transmitted homologs to the HGT candidates during the infection process. The transcriptional profiling experiment was conducted at three important time points during the infection process: early , middle  and late . Candidates GLRG_11091 (HGT2) and GLRG_11966 (HGT2) of C. graminicola and candidates CH063_02340 (HGT2), CH063_10640 (HGT2) and CH063_01625 (HGT5) of C. higginsianum are strongly upregulated at the PA and BP time points which are very early in the infection process, suggesting a role in plant penetration or establishment of infection .From Figure Colletotrichum. The construction and analysis of phylogenetic trees is recognized as the most reliable method for detecting HGT, so we included several steps of manual phylogenetic trees inspection in the pipeline. Using this pipeline, we detected 12 genes with evidence of HGT from bacteria and one from plants to the genus Colletotrichum. The genes were classified into 11 families of homologous sequences  suggests a different role in this pathogen. In another study, a mutant of ARG1, an arginosuccinate lyase encoding gene in Fusarium oxysporum f. sp. melonis showed reduced virulence [Many of the HGT candidates are involved in metabolic processes such as synthesis of amino acids , lipid metabolism (HGT3), sugar metabolism  and secondary metabolism HGT2). HGT1 belongs to a family of argininosuccinate lyase genes responsible for the formation of arginine and fumarate in the urea cycle. In . HGT1 bea et al.  found thirulence . These sColletotrichum and HGT11 is found in Colletotrichum as well as the closely related genus Verticillium. In bacteria, the horizontal transfer of amino acid metabolic genes have been proposed to provide metabolic plasticity to the recipient species of both non pathogenic [Other enzymes with roles amino acids metabolism are the members of HGT7, a family of L-asparaginases in the aspartic acid synthesis pathway and HGT11 a family of succinyl-diaminopimelate desuccinylase enzymes in the lysine biosynthesis pathway. HGT7 was found only in the genus thogenic , 55 and thogenic  species,thogenic , 57.Many of the HGT candidates have functions related to sugar and lipid metabolism , consistent previous reports . RichardC. graminicola  of C. graminicola does not have changes in expression levels during the infection process, suggesting that its role during infection may be different from that of its C. higginsianum counterpart.The putative functions of genes in HGT4 and HGT6 in the degradation of cell wall sugars (HGT4) and proteins (HGT6) and the presence of a predicted secretion signal peptide opens the possibility that they may be secreted lytic enzymes. The time of overexpression of GLRG_11936 (HGT6) in l et al.  in the tCordyceps militaris and Beauveria bassiana. This suggests that these genes have an important function in host-fungal interactions and that it has maintained this function for millions of years.The HGT events HGT4 and HGT6 are among the most ancient, having occurred at least 397 million years ago. The transferred genes have been retained in only a few species, all of which are pathogenic, and in the case of HGT6, includes the entomopathogens A large number of the HGT candidates have functions related to amino acid and carbohydrate metabolism. In the Ascomycetes, gene families with these functions were associated with “volatility”, a term introduced by Wapinsky et al.  to descrWe also developed a time calibrated phylogenetic tree and deduced the ages of the HGT events Figure . It is iColletotrichum HGT candidates, reflecting the propensity of gene loss in horizontally transferred genes. The molecular clock analysis reveals that the HGT events detected in the present have an ancient origin. The HGT candidates typically belong to volatile gene families that are subject to frequent gene duplication and loss. Nevertheless, it is possible that the horizontally transferred gene provided new functions that were useful to ancestral fungi, enabling them to colonize new niches or improve fitness. With the evidence in this work and in other studies, we propose that HGT has been an important evolutionary force in the Pezizomycotina.The phylogenetic evidence presented in this work shows that the genetic flux from bacteria to Pezizomycotina have been constant. Most of the Pezizomycotina with complete genomes available have no evidence of homologs to the http://www.uniprot.org). Python scripts were used to extract the sequence, description and the two highest taxonomic levels of each sequence. The two highest taxonomic levels of UniProt correspond to kingdom and phylum in Archaea and Bacteria and to superkingdom and kingdom in Eukaryota. The two highest levels were selected to identify the taxonomic assignment at the level of kingdom of the sequences in the database. With this database a BLASTP (v2.2.29+) [-5) was performed with all putative proteins predicted in the genomes of C. graminicola M 1001, C. higginsianum IMI 349063 [C. gloeosporioides[The first BLAST search was performed using a database of proteins from organisms with complete proteome available in UniProt (downloaded 19-12-2011) (2.2.29+)  search  (476 proteins from the 3 species were selected). For the second phylogeny we performed a BLAST search of the GenBank nr database (downloaded 03-02-2012) and included the 20 best hits from each kingdom  to avoid a possible under or overrepresentation for the abundance of sequences from any one kingdom in the BLAST results. The sixty best BLAST hits were aligned using MAFFT  and the The third phylogeny was constructed with the best 100 BLAST hits from the nr database (downloaded 20-05-2012). The phylogenetic trees constructed as described in the second phylogeny and were evaluated manually, selecting only those that have well-supported topologies that are clearly concruent with HGT. Asymmetric or ladder-shape trees were excluded because such tree topologies are often a signal of long branch attraction or lack of phylogenic information . AdditioC. graminicola, C. higginsianum and C. gloeosporioides genomes using BLAST (v2.2.29+). When no evidence of homology in one or more species was found, we additionally searched the EST database of NCBI and the RNA-seq sequences of O’Connell et al. [C. graminicola, BLAST searches were performed in the genome of 5 sequenced strains of this species by Rech [Colletotrichum genomes were used to verify the absence/presence of each candidate in other members of the genus. This verification was performed through BLAST searches. The genomes used for this purpose were: Colletotrichum gloeosporioides 23  (http://www.jgi.doe.gov), Colletotrichum fiorinae MH 18  (http://www.jgi.doe.gov), Colletotrichum gloeosporioides Nara gc5 [Colletotrichum orbiculare MAFF 240422 [Homologs to the HGT candidates were identified in the l et al.  to find  by Rech . AdditioNara gc5  and CollF 240422 .The 11 groups of candidates were subjected to three tests to ensure that there is sufficient phylogenetic signal to support the HGT hypothesis. For each candidate, we manually inspected the results of the BLAST search to the nr database (downloaded 12-11-2012), selecting appropriate percent identity and coverage thresholds to eliminate distantly related and poorly aligning homologs. Each HGT candidate and its homologs were aligned and analyzed with TREE-PUZZLE . The TREWe evaluated the stability of the tree topology when different sets of proteins are used. For each candidate we reconstructed several trees by randomly removing several proteins and comparing the resulting tree topology with that of the original. HGT2, HGT6 and HGT3 had different topologies in this analysis but were stable in the rest of the tests to evaluate the phylogenies.The data sets were used to perform topology tests to determine whether a tree toplogy supporting vertical inheritance or a star phylogeny are equally well supported by the sequence alignments. Some of the candidates did not have high similarity with fungal sequences or sequences of any other kingdom more than bacteria and in these cases the topology tests could not be performed because the putative HGT sequences could not be constrained with the fungal branch in the tree to evaluate vertical inheritance as an alternative hypothesis. To generate the tree consistent with vertical inheritance, MrBayes  was usedLaccaria bicolor. However, the most similar BLAST hits are from bacteria . Therefore, we conclude that this is neither a case of mitochondrion to nucleus transfer nor an error in the genome assembly.To determine whether any of the HGT candidates are mitochondrial genes that were incorrectly assembled into the nuclear genome sequence, or are mitochondrion to nuclear HGT events, we performed a BLASTP (v2.2.29+) search vs all mitochondrial proteins available in the RefSeq database . ProteinC. graminicola, C. gloeosporioides and C. higginsianum. Most of the neighboring genes showed the expected distribution of BLAST hits with Colletotrichum and fungal proteins  search of the nr database of NCBI (online version 06-03-2013) was performed with the upstream and downstream proteins of each HGT candidate in the genome of 3) searches were conducted with four chains each (three heated and one cold). The convergence between them was checked using a sample frequency of 1000 generations. A burn-in of 25% of generations was excluded to reconstruct the Bayesian consensus tree. The topology differences between maximum likelihood and Bayesian trees were quantified with the ratio of identical nodes performed in T-coffee [A maximum posterior tree was constructed with MrBayes, performing 2,000,000 generations of samples, using the substitution matrix and model predicted by MODELGENERATOR but allowing the program to calculate the proportion of invariable sites and the alpha parameter for gamma distribution. Two Multiple Chain Markov Chain Monte Carlo  search against all mitochondrion proteins of RefSeq database [To determine if HGT candidates were the result of contamination a BLASTP (v2.2.29+) (vs the nr database of NCBI) search of the upstream and downstream protein of each candidate in the genome of database  was perfdatabase  to detecThe program Blast2GO 2.8  was usedBLASTP (v2.2.29+) searches of the HGT candidates were performed against PHI-base V3.4 , VirulenC. graminicola and C. higginsianum. The data is presented in the Additional file The expression data were extracted from O’Connell et al.  for C. gC. graminicola a Mann–Whitney-Wilcoxon test was performed using R v2.13.1 [C. graminicola gene was calculated from the information available on the Broad institute website (http://www.broadinstitute.org).To determine the presence/absence of introns in HGT candidates a manual inspection of genes was made. To compare the difference of introns content among the candidates and  v2.13.1 . The intColletotrichum genome and HGT candidates. These indices were used to calculate the differences of GC content among candidates and whole genomes.The program CodonW  was usedhttp://www.uniprot.org), GenBank (http://www.ncbi.nlm.nih.gov/genbank/), Joint genome institute (http://genome.jgi-psf.org/programs/fungi/index.jsf?projectList) and Broad Institute (http://www.broadinstitute.org/) databases . To reconstruct the species tree, the amino acids inferred from six nuclear genes were chosen. The proteins selected  from FunyBase [To estimate the phylogenetic relationship of the species involved in HGT events, all fungal species found in BLAST searches of the HGT candidates were used . The complete proteome of these species was obtained from the UniProt  as the lower bound for the Pezizomycotina crown [http://www.tree.bio.ed.ac.uk/software/figtree/).To estimate the putative age of the transferred genes a fossil calibrated molecular clock analysis was performed. To avoid problems of convergence in the calculations of the calibrated tree, we selected 15 species from the 54 taxa used in the species tree. The same matrix of 6 concatenated proteins of the species tree was used. The analysis was performed with the BEAST v1.7.5 software package . For eacna crown . A lognona crown ), standana crown  and the na crown  dismissina crown . The resBLAST hits of neighboring genes of HGT candidates.Table S3. Number of gene losses of the HGT in different Pezizomycotina lineages, based on whole genome sequences available in GenBank. Table S4. List of the species involved in the HGT events. Figure S1. The pipeline used to detect HGT candidates. Figure S2. Maximum likelihood tree of HGT2. Figure S3. Maximum likelihood tree of HGT3. Figure S4. Maximum likelihood tree of HGT4 Figure S5. Maximum likelihood tree of HGT5. Figure S6. Maximum likelihood tree of HGT6. Figure S7. Maximum likelihood tree of HGT7 Figure S8. Maximum likelihood tree of HGT8. Figure S9. Maximum likelihood tree of HGT9. Figure S10. Maximum likelihood tree of HGT10. Figure S11. Maximum likelihood tree of HGT11. (PDF 4 MB)Additional file 1:C. graminicola and C. higginsianum. (XLSX 42 KB)Additional file 2: Table S2: Expression of HGT candidates and homologs in


Mycoplasma hyopneumoniae is the cause of enzootic pneumonia in pigs, a chronic respiratory disease associated with significant economic losses to swine producers worldwide. The molecular pathogenesis of infection is poorly understood due to the lack of genetic tools to allow manipulation of the organism and more generally for the Mycoplasma genus. The objective of this study was to develop a system for generating random transposon insertion mutants in M. hyopneumoniae that could prove a powerful tool in enabling the pathogenesis of infection to be unraveled. A novel delivery vector was constructed containing a hyperactive C9 mutant of the Himar1 transposase along with a mini transposon containing the tetracycline resistance cassette, tetM. M. hyopneumoniae strain 232 was electroporated with the construct and tetM-expressing transformants selected on agar containing tetracycline. Individual transformants contained single transposon insertions that were stable upon serial passages in broth medium. The insertion sites of 44 individual transformants were determined and confirmed disruption of several M. hyopneumoniae genes. A large pool of over 10 000 mutants was generated that should allow saturation of the M. hyopneumoniae strain 232 genome. This is the first time that transposon mutagenesis has been demonstrated in this important pathogen and could be generally applied for other Mycoplasma species that are intractable to genetic manipulation. The ability to generate random mutant libraries is a powerful tool in the further study of the pathogenesis of this important swine pathogen. Mollicutes, mycoplasmas are characterised by their lack of a cell wall and small genome size, and are considered to be the smallest free-living self-replicating organisms and as such are of considerable interest in synthetic biology  such that the C9 mutant Himar1 is under control of the P97 promoter sequence.PCR was performed using a Phusion High Fidelity kit  according to the manufacturer’s instructions. Sequences of oligonucleotides used in the study are shown in Table id pSRT2  was ampl plasmid . Mariner plasmid . To consin pSRT2  was ampltrain 232 genomic 29b + C9 , adding M. hyopneumoniae strain 232 [2. For the selection of transformants, tetracycline hydrochloride  was added to Friis broth medium and Friis agar medium at final concentrations of 0.5 μg/mL and 0.2 μg/mL respectively. Molecular cloning was performed using Escherichia coli strain DH5α, grown in Luria-Bertani medium according to standard methods [tetM gene, tetracycline hydrochloride was added to a final concentration of 5 μg/mL.rain 232  was growrain 232 . For gro methods . TransfoM. hyopneumoniae strain 232 was grown to mid-late logarithmic phase in Friis broth as determined by an acid colour change in the phenol red pH indicator. Mycoplasmas were harvested by centrifugation of culture at 9000 × g for 10 min at 4 °C and washed three times in electroporation buffer . One hundred microliters of cells (corresponding to 3 mL culture) were incubated on ice with approximately 10 μg plasmid DNA for 30 min. Electroporation was performed in a 0.2 cm cuvette  at 2.5 kV, 100 Ω, 25 μF and 900 μL ice-cold Friis medium were immediately added. After 15 min incubation on ice, cells were transferred to a 1.5 mL tube and incubated at 37 °C for 3 h. Culture was then plated onto Friis agar containing tetracycline and incubated at 37 °C in 5% CO2 for up to 18 days. Individual tetracycline-resistant transformants were picked using a sterile pipette tip into Friis broth medium containing tetracycline and grown for 5–7 days until evidence of growth as determined by the pH indicator.M. hyopneumoniae strain 232 culture using a phenol-chloroform method [tetM and plasmid backbone was determined by PCR using primer pairs TetMF/TetMR and Tpn1/Tpn2 respectively. Two and a half micrograms of DNA were digested overnight at 37 °C with 10 units of AluI, which cuts the 892 kbp genome of M. hyopneumoniae strain 232 a total of 2239 times and generates blunt ends. AluI also cuts within the tetM transposon sequence six times. The digested DNA was purified using a MinElute PCR Purification kit . Oligonucleotide linkers were attached to the digested DNA fragments. To generate the linker, 10 μM oligonucleotides Linker-A and Linker-B  for 1 h at room temperature. The ligated DNA was purified using a MinElute PCR Purification kit . PCR was performed using HotStarTaq  according to the manufacturer’s instructions. Thermocycler conditions were as follows: 95 °C for 15 min; 33 cycles of 94 °C for 45 s, 55 °C for 60 s, 72 °C for 120 s; 72 °C for 10 min. PCR products were visualised by electrophoresis in 1.6% agarose. Transposon insertion sites were determined by the direct sequencing of PCR products.Transposon insertion site determination was based on the method described by Chaudhuri et al. . GenomicHindIII. DNA was separated by electrophoresis on 0.9% agarose, blotted onto Hybond-N + membrane  and then fixed to the membrane by exposure to UV light. A digoxigenin (DIG)-labeled probe specific for the tetM gene was generated from PCR-amplified DNA  strain and a small number of transposon insertions in M. hyopneumoniae strain 232 [4001 and a transposon carrying the pac gene conferring resistance to puromycin  were required after transformation prior to selection of transformants on solid medium. We attempted to transform M. hyopneumoniae with pMiniHimar1BSC1 and using a self-replicating oriC plasmid (pMHOpuro) as a positive control for transformation [6 CFU were transformed with pMiniHimar1BSC1 and with pMHOpuro. On this occasion, no puromycin resistant transformants were generated with plasmid pMiniHimar1BSC1 while an average of 1.5 × 10-4 transformants/CFU were produced by the pMHOpuro positive control. To determine whether tetM selection would increase the yield of transformants, pac was replaced with tetM in plasmid pMHWT-2 . Approximately 106 CFU were transformed with pMHWT-2, pMHWT-1, pMHC9-1 and pMHO-2 and selection plates examined after incubation for 14 days. Plasmid pMHWT-2 failed to generate any tetracycline resistant colonies compared to an average of 4.6 × 10-5 transformants/CFU for the pMHO-2 positive control Table 8 CFU per transformation. Again, more transformants were generated with the hyperactive transposase (33 per transformation) compared to the wild-type transposase (10 per transformation). Transformation of an increased number of cells resulted in a drop in the overall transformation efficiency. However, our experiments suggested that the hyperactive C9 mutant Himar1 transposase could account for a 3-4-fold increase in transformation efficiency compared to the wild-type transposase. Morphologically, tetracycline-resistant colonies grown on Friis agar from transformed cells had a uniform appearance that resembled that of colonies grown from non-transformed cells  was recently described that successfully generated transposon insertion mutants in the rain 232 . pMiniHiormation . Approxirain 232 . Plasmidrain 232 . AdditioM. hyopneumoniae strain 232 with plasmid pMHC9-1 consistently generated tetracycline resistant colonies on Friis agar containing tetracycline. To confirm that tetracycline resistance was due to transposition into the host chromosome and not, for example, due to maintenance of extrachromosomal plasmid DNA, 15 individual colonies from pMHC9-1 transformed M. hyopneumoniae strain 232 were analysed. Each was transferred into 1 mL Friis broth medium containing 0.5 μg/mL tetracycline. All transformants grew within 5 days, confirming the maintenance of tetracycline resistance upon sub-culturing into liquid medium while no growth was observed for untransformed controls. PCR amplification of tetM from total DNA extracts prepared from 20 mL culture confirmed the presence of tetM in all transformants generated with the hyperactive C9 mutant transposase but not un-transformed M. hyopneumoniae strain 232  and 5 non-coding regions (NCRs). For 5 ORFs, there was more than one transformant containing an insertion. The average size of an ORF in the M. hyopneumoniae genome is 388 amino acids (aa). All 5 of these ORFs were larger than 388 aa and it is therefore possible that more than one transformant was generated due to the larger size of these ORFs. However, a total of 11 transformants contained a transposon insertion in mhp447. Although this ORF is particularly large at 3970 aa, representing 1.3% of the M. hyopneumoniae strain 232 genome, it is likely that this represents a “hotspot” for transposon insertions. Similarly, 4 transformants contained insertions in the smaller pepF gene (608 aa) and this too may be over-represented. Most of the transposon insertions are in genes of unknown function but that appear to be dispensable for axenic growth, or in NCRs. Of the insertions in genes of known function, three were in the P97 ciliary adhesin and the P95 outer membrane protein. Insertions were found in 3 genes encoding phospotransferase system subunit proteins specific for fructose and ascorbate and in the molecular chaperone Trigger Factor. Several insertions were also identified in the pepF gene encoding oligoendopeptidase F and the pepP gene encoding a proline aminopeptidase. These are two of a small number of proteases that may be associated with processing of M. hyopneumoniae cell surface proteins [Insertion site sequencing of a further 33 individual transformants was performed so that the locations of a total of 44 transformants were mapped to the e Figure . Of thesM. hyopneumoniae. A pool of at least 10 000 individual mutants would be expected to give a very high probability of an insertion into every one of the 692 protein coding sequences in the M. hyopneumoniae 232 genome determined by Minion et al. [6 CFU per transformation. After 14 days growth at 37 °C on agar plates containing tetracycline, colonies were counted and harvested together in one large pool. A total of 11 759 colonies were counted across all 20 plates, representing an average transformation frequency of 3.4 × 10-4 transformants/CFU (standard error 5.3 × 10-5).We sought to determine whether our pMHC9-1 transposon delivery vector could be used to produce a large pool of random mutants, sufficient to provide an adequate number of “hits” in all of the non-essential genes of n et al. . It was M. hyopneumoniae has stood as a hurdle to advancements in the understanding of the pathogenesis of enzootic pneumonia in pigs. We recently reported the successful transformation of M. hyopneumoniae using an artificial plasmid system containing the origin of replication of M. hyopneumoniae and an antimicrobial resistance cassette, that was capable of self-replicating and maintenance in transformed bacteria [M. hyopneumoniae by electroporation with plasmid DNA. This knowledge has now enabled the design and construction of a transposon-based system for generating random mutants in M. hyopneumoniae.The inability to efficiently genetically manipulate bacteria . We have4001 transposon is effective in generating insertional mutations in a number of different Mycoplasma species [M. hyopneumoniae. A Himar1 transposon delivery vector that was shown to be active in M. gallisepticum generated very low numbers of transformants in M. hyopneumoniae with a very restricted range of unique insertion sites [M. hyopneumoniae strain 232-specific promoter sequence to control the Himar1 expression and switching the wild-type Himar1 transposase for a hyperactive mutant C9 form, the frequency of transposition increased significantly. Analysis of transformants confirmed that resistance to tetracycline was due to a transposition event into the mycoplasma genome and it appeared that transposition occurred at a single site in each individual transformant. The transposon insertions appeared to be stable over serial passages and were maintained even in the absence of antimicrobial selection. These features are desirable when the phenotypes of mutants are studied in vitro, and particularly for in vivo studies of pools of mutants by TraDIS or signature-tagged mutagenesis. Additionally, we did not encounter any “pseudoresistant” colonies in our “no DNA” control transformations, which have been documented in transposon mutagenesis systems described for other mycoplasmas [The Tn species ,18,19,34on sites . By usinoplasmas ,26. “PseM. hyopneumoniae is assessed and monitored by a simple change in the phenol red pH indicator incorporated into the medium, owing to the fact that turbidity is not generated. Thus pH can serve as a rough guide for growth phase, but the actual number of cells can only be estimated after plating the culture out and allowing growth on agar medium for 5–7 days. Additionally, complex and undefined medium is used to grow M. hyopneumoniae, with 20% equine/porcine serum and freshly prepared yeast extract contributing to variations in batches of medium.In the course of this study, it was evident that there were significant differences in the transformation frequencies obtained using plasmid pMHC9-1. We suspect that differences in transformation frequency can be partially accounted for by variations in the number of cells electroporated and/or the growth phase of the mycoplasma culture used. It appeared that an increase in the number of cells transformed did not necessarily result in an increase in transformation frequency. However, even where similar numbers of cells were electroporated, there could be significant differences in transformation frequencies, implying that other factors could be important. Growth of M. hyopneumoniae genes of both known and unknown function, and in non-coding regions. We observed a clustering of transposon insertions in mhp447, a large open-reading frame of unknown function. The Himar1 transposon inserts at 5’-TA-3’ dinucleotides and we considered that the large number of insertions may be due to an increase in AT content. However, analysis of this ORF showed that the AT content was not unusual at 0.681 compared to 0.714 for the entire M. hyopneumoniae strain 232 genome. Alternatively, it is possible that there are hotspots for Himar1 insertions in the M. hyopneumoniae strain 232 genome, that may be affected by factors such as DNA topology, as is the case with other transposons, including Mariner elements [M. hyopneumoniae. This pool of mutants may prove a valuable tool in future studies of M. hyopneumoniae in allowing a list of minimal essential genes to be determined. Such a pool of mutants could also be subjected to an in vivo screen for attenuated mutants using powerful functional genomic techniques such as TraDIS.Transposon insertion sites were determined for a limited number of individual transformants using a linker PCR technique and direct sequencing of PCR products by Sanger sequencing. From our limited analysis, we found transposon insertions in a number of elements . It is dM. hyopneumoniae, allowing for the first time the production of large numbers of random transposon insertion mutants. This system has been used to generate mutants with random transposon insertions in individual M. hyopneumoniae protein coding and non-coding sequences. It has also been used to generate a large pool of over 10 000 transposon insertions. These mutants are powerful tools in elucidating the functions of M. hyopneumoniae genes, and in turn unraveling the pathogenesis of this important swine pathogen.A novel transposon delivery system has been generated for A patent application covering the method of transposon mutagenesis described in this paper has been filed by the Royal Veterinary College. The authors declare that they have no further competing interests.Experiments were conceived by GAM, ANR and BSC. Experiments were performed by GAM and ASD. BSC, JTB, ANR, SEP, PRL, BWW, AWT and DJM assisted with experimental design. The manuscript was written by GAM and ANR and was approved by all authors.BRaDP1T consortium. List of researchers collaborating in the BRaDP1T consortium.Click here for file


CDH23 mutations in East Asians, its large size hinders investigation. The pathologic mutation p.P240L in CDH23 is common in East Asians. However, whether this mutation represents a common founder or a mutational hot spot is unclear. The prevalence of CDH23 mutations with prelingual severe-to-profound sporadic or autosomal recessive sensorineural hearing loss (arSNHL) is unknown in Koreans.Despite the prevalence of GJB2, GJB6 and GJB3. Sanger sequencing of CDH23 p.P240L was performed on connexin-negative samples without enlarged vestibular aqueducts (EVA), followed by targeted resequencing of 129 deafness genes, including CDH23, unless p.P240L homozygotes were detected in the first screening. Four p.P240L-allele-linked STR markers were genotyped in 40 normal-hearing control subjects, and the p.P240L carriers in the hearing-impaired cohort, to identify the haplotypes.From September 2010 to October 2014, children with severe-to-profound sporadic or arSNHL without phenotypic markers, and their families, were tested for mutations in connexins CDH23 mutant alleles, and SLC26A4 and GJB2 accounted for 18.0 and 17.2 %, respectively. All four children showed profound nonsyndromic SNHL with minimal residual hearing. Interestingly, all had at least one p.P240L mutant allele. Analysis of p.P240L-linked STR markers in these children and other postlingual hearing-impaired adults carrying p.P240L revealed that p.P240L was mainly carried on a single haplotype.Four (3.1 %) of 128 children carried two GJB2-negative arSNHL Koreans without EVA is recommended.p.P240L contributed significantly to Korean pediatric severe arSNHL with a strong founder effect, with implications for future phylogenetic studies. Screening for p.P240L as a first step in The online version of this article (doi:10.1186/s12967-015-0624-8) contains supplementary material, which is available to authorized users. Many loci can cause genetic sensorineural hearing loss (SNHL), which accounts for more than 50 % of SNHL . New molCDH23 (NM_022124) is a prime example as this gene consists of 69 exons, 11,073 bases, and is located on chromosome 10q21–q22 ) resulting in autosomal recessive hearing loss with negative GJB2 and SLC26A4 mutations [The advent of NGS rapidly led to identification of pulation , 14–16. in Japan . Recentlin Japan  reportedpatterns . Anotherutations . Most ofutations , 19.CDH23 remains expensive and not cost-effective. Nevertheless, p.P240L, a predominant CDH23 mutation in Japanese and Koreans, is of interest as it accounts for ~43.3 % and >50 % of total CDH23 mutations, respectively [Rigorous and costly bioinformatic analyses are needed to screen for these mutations, and despite significant reductions in the cost of some NGS techniques, routine clinical screening for ectively , 18, 19.CDH23 mutation to nonsyndromic arSNHL was estimated in a Korean pediatric hearing loss cohort, and the genetic load of GJB2 and SLC26A4 was documented. Moreover, whether p.P240L, the most common CDH23 mutation in Koreans, represented a common disease-associated founder mutation was investigated. The clinical characteristics  of the hearing-impaired subjects carrying CDH23 mutations were also documented.In this study, the relative genetic contribution of the This study was approved by the Institutional Review Boards (IRBs) at the Seoul National University Bundang Hospital (SNUBH) (IRB-B-1007-105-402) and the Seoul National University Hospital (SNUH) (IRBY-H-0905-041-281). Written informed consent was obtained from all participating subjects. In case of the children, written informed consent was obtained from their parents or guardians on their behalf.A total of 438 subjects who visited hereditary hearing loss clinics of tertiary referral centers: (SNUH and SNUBH) for their SNHL from September 2010 to October 2014 were initially enrolled in this study. Clinical evaluations were performed in this cohort, including gender, date of birth, medical history, physical examination, pure tone audiometry and when available, imaging studies .All of the enrolled subjects underwent an audiologic evaluation. The pure-tone thresholds were recorded at 0.25, 0.5, 1, 2, 4 and 8 kHz. However, some infants could be recorded at only limited frequencies due to poor cooperation. The hearing threshold was calculated by averaging the thresholds of 0.5, 1, 2 and 4 kHz, and was classified as subtle (16–25 dB), mild (26–40 dB), moderate (41–70 dB), severe (71–95 dB), or profound (>95 dB).CDH23 mutation frequency with those of mutations in SLC26A4 and other deafness genes in the same cohort  were included to compare the ort Fig. . ConsequCDH23 and calculation of the normal carrier frequency of p.P240L, respectively.We recruited two control groups: the control group I included randomly chosen 40 Korean adult subjects with pure tone audiograms indicating normal hearing thresholds and the control group II comprised independent 272 adult subjects with available whole exome sequencing data and with allegedly normal hearing. In this control group II, whole exome sequencing was performed prior to this study to make a molecular diagnosis of diseases irrelevant to SNHL and their hearing status was evaluated by the interview. The control group I and II was recruited for the analysis on the haplotypes around p.P240 of SLC26A4. Sanger sequencing of GJB2, 3 and 6 was performed on subjects without noticeable phenotypic markers, as described previously [Molecular genetic studies were conducted in 128 probands and their family members. Genomic DNA was extracted from peripheral blood using standard protocols . The nonsyndromic prelingual arSNHL subjects with an enlarged vestibular aqueduct (EVA) underwent direct PCR-based Sanger sequencing of eviously  GJB2, GJB3, and GJB6 were subject to Sanger sequencing of p.P240L, which was the most frequent mutant allele of CDH23 in Korean cochlear implantees, as reported in our previous study [CDH23 was CDH23-8F(5′-CCACCATCACTCAACCTAAG-3′) and CDH23-8R (5′-ACTAACAGGGCCCATCCCTA-3). Next, targeted resequencing of 80, 129  or 200 known deafness genes [CDH23, was performed on the remaining non-EVA samples without a conclusive molecular diagnosis after sequencing of GJB2, GJB3, and GJB6, as described previously [CDH23 variants in a trans configuration.Non-EVA samples that were also negative for mutations in connexins us study  Fig. 1)GJB2, GJBss genes  includineviously , 21, witCDH23 in Koreans was determined to ascertain whether the mutant allele was a common founder. For this purpose, we chose four STR markers flanking the CDH23 gene  in total, segregating p.P240L of CDH23, and also in 40 adult Korean subjects with documented normal hearing thresholds.One multiplex family (SB116) with co-segregating post-lingual onset of SNHL and a heterozygous p.P240L mutant allele of The allele frequency of p.P240L was compared between prelingually onset, severe-to-profound sporadic/autosomal recessive SNHL in a pediatric population and 272 normal hearing controls (control group II). Differences in STR genotypes and the distribution of haplotypes between mutant chromosomes carrying p.P240L and 80 wild-type Korean chromosomes from 40 normal-hearing subjects (control group I) were analyzed by Fisher’s exact test. One postlingual adult DFNB12 subject (SB116-208) was additionally included for the STR genotyping, while the subject was excluded for calculation of the allele frequency of p.P240L among pediatric prelingual severe to profound SNHL. Contributions of some genotypes were compared among subjects, but not between chromosomes, due to the lack of parental samples, as described previously , 23. Espp value of <0.05.Statistical analyses were performed using SPSS 18.0 . The level of statistical significance was defined as a GJB2 (DFNB1) and 23 (18.0 %) carried two mutant alleles of SLC26A4 (DFNB4). The detection frequency of CDH23 mutations  had two mutant alleles of .2 % had Interestingly, all four subjects carried p.P240L as either a homozygote or compound heterozygotes, suggesting that this mutant allele was either a strong common founder or a mutational hotspot in this population Table  Fig. 3)3).Table To ascertain whether p.P240L was a causative variant in these subjects, the carrier frequency of the p.P240L allele was calculated among 272 adult controls with allegedly normal hearing thresholds (control group II). None of the normal control subjects carried the mutant allele, which further confirmed its pathogenic potential   were constructed and analyzed in five unrelated probands from five families , and in 40 adult Korean normal-hearing subjects (control group I) Table ; Fig. 2bCDH23 mutations in our pediatric cohort with severe-to-profound nonsyndromic arSNHL was 3.1 %. This was in agreement with findings for CDH23 variants in other studies on East Asian populations, the prevalences of which ranged from 1.6 to 15.4 %, using cohorts with nonsyndromic arSNHL not necessarily limited to prelingual pediatric deaf subjects (Table CDH23 mutations (15.4 vs 3.1 %) obtained from a different Korean cohort with nonsyndromic arSNHL not limited to prelingual SNHL cases [CDH23 mutations also significantly contribute to adult-onset postlingual arSNHL in Koreans. Indeed, in contrast to the p.P240L mutation, which presented mostly as prelingual severe-to-profound hearing loss, certain CDH23 mutations, such as p.R2029W and p.T1368M, were reported to be associated with postlingual onset of moderate hearing loss, and most CDH23-affected subjects showed progressive hearing loss [The prevalence of recessive HL cases   phenotype is more commonly associated with CDH23 mutations than is the syndromic hearing loss phenotype (USH1D).The p.P240L allele can be classified as a DFNB12 allele, rather than an USH1D allele, based on a previous hypothesis  and the GJB2 mutations in some Western cultures can be attributed to a single ancestor mutation [TMPRSS3 affects a wide ethnic spectrum, encompassing East Asians and Europeans, and hence is predicted to be a mutational hot spot [GJB2 and the H723R and IVS7-2A>G mutations of SLC26A4, is restricted to East Asians [CDH23 mutations and the haplotypes in other ethnic groups would enable tracing of the common ancestor or route of divergence of the CDH23 mutation. This information would have phylogenetic applications.The geographic or ethnic spectra of founder mutations vary, and whether the most frequent mutation allele represents a founder mutation and/or a mutational hot spot is often unclear. Some founder mutations have a narrow distribution; for example, due to the practice of consanguineous marriage, the high prevalence of mutation . On the hot spot , 32, 33.t Asians , 30. Onlt Asians , 19, 34,t Asians , 6, 14. t Asians , 36. FurCDH23 mutations in Korean children with prelingual onset, nonsyndromic, sporadic or autosomal recessive, severe-to-profound SNHL was 3.1 %. p.P240L was the most common CDH23 mutation. Genotyping of STR markers linked to p.P240L showed that the p.P240L mutation had a strong founder effect in Koreans. This novel finding may facilitate genetic diagnosis of, and research into, CDH23 mutations, as well as provide information for further phylogenetic studies.The genetic load of


Treacher Collins syndrome  is a rare congenital disorder of craniofacial development. Characteristic hypoplastic malformations of the ears, zygomatic arch, mandible and pharynx have been described in detail. However, reports on the impact of these malformations on speech are few. Exploring speech features and investigating if speech function is related to phenotypic severity are essential for optimizing follow-up and treatment.Articulation, nasal resonance, voice and intelligibility were examined in 19 individuals  divided into three groups comprising children 5–10 years (n = 4), adolescents 11–18 years (n = 4) and adults 29 years and older (n = 11). A speech composite score (0–6) was calculated to reflect the variability of speech deviations. TCS severity scores of phenotypic expression and total scores of Nordic Orofacial Test-Screening (NOT-S) measuring orofacial dysfunction were used in analyses of correlation with speech characteristics (speech composite scores).Children and adolescents presented with significantly higher speech composite scores  than adults . Nearly all children and adolescents (6/8) displayed speech deviations of articulation, nasal resonance and voice, while only three adults were identified with multiple speech aberrations. The variability of speech dysfunction in TCS was exhibited by individual combinations of speech deviations in 13/19 participants. The speech composite scores correlated with TCS severity scores and NOT-S total scores. Speech composite scores higher than 4 were associated with cleft palate. The percent of intelligible words in connected speech was significantly lower in children and adolescents  than in adults . Intelligibility of speech among the children was markedly inconsistent and clearly affecting the understandability.Multiple speech deviations were identified in children, adolescents and a subgroup of adults with TCS. Only children displayed markedly reduced intelligibility. Speech was significantly correlated with phenotypic severity of TCS and orofacial dysfunction. Follow-up and treatment of speech should still be focused on young patients, but some adults with TCS seem to require continuing speech and language pathology services. Treacher Collins syndrome  is a rare congenital disorder of craniofacial development affecting 1 in 50 000 live births . TCOF1 mThe variability of phenotypic expression of TCS is considerable . The chiIsolated cleft palate has been reported in approximately one-third of affected individuals ,9,11. OtAnterior open bite and Class II malocclusion are common findings in children and adolescents with TCS, while Class I and III malocclusion are more rare ,11. OrthVariable degrees of auricular deformities and malformations of the middle ear are typical features of TCS ,12. AtreA combined retrospective and prospective study of speech comprising 30 children and adolescents affected with TCS has revealed articulation errors in all participants . The artAnother study has reported speech abnormalities in 74 percent of the 46 patients with TCS who were treated at a craniofacial unit from birth to maturity . The abnPrevious studies have established that speech deviations are common in young individuals with TCS. However, there are to our knowledge no data on speech in adults or any evaluations of intelligibility in order to measure how well a listener is able to map the acoustic signal onto the intended lexical units . FurtherThe objectives of the present study were to explore speech features associated with TCS, and to investigate how these speech characteristics are related to phenotypic severity of the condition.The specific questions of the present study were:• How similar are the speech features in TCS in terms of articulation, nasal resonance and velopharyngeal function, voice and intelligibility across ages?• Are speech characteristics, measured as a speech composite score extracted from findings of articulation errors, nasal resonance and voice aberrations, correlated with phenotypic severity and orofacial function?• How are structural malformations, like malocclusion, cleft palate and hearing loss, associated with articulation errors, nasal resonance, velopharyngeal dysfunction, altered voice and intelligibility in TCS?All patients with TCS registered at either the TAKO-centre , the Department of Medical Genetics and the Centre of Rare Medical Conditions based at Oslo University Hospital, or the Norwegian Craniofacial Association (patient support group), were contacted by mail and offered an extensive health examination focusing on orofacial characteristics and functions including assessment of speech. Twenty-three of the 36 eligible individuals accepted the invitation and all gave informed consent to participate in the present study. Informed consent was obtained from parents of participants below 16 years.The recruitment procedures and study protocol were approved by the Regional committee for medical research ethics at the University of Oslo  as well as the Norwegian Data Inspectorate at Oslo University Hospital .The study group comprised 19 individuals from 14 families, 13 females and 6 males, geographically spread over ten of the nineteen Norwegian counties. The median age was 34 years (range 5–74). None of the participants fell within the age range 19–29 years. The participants were divided into three subgroups due to potential influence of speech developmental factors: children 5–10 years (n = 4), adolescents 11–18 years (n = 4) and adults from 29 years and older (n = 11). The TCS diagnosis was confirmed by a clinical geneticist . Four ofAll data were collected within a period of six months. The participants attended clinical examinations during two consecutive days and all assessments were carried out by the same specialists.Data on type of cleft including reconstruction with pharyngoplasty, atresia of the ear canal, hearing, nasal obstruction, narrow hypopharynx including history of tracheostomy, malocclusion, orthodontic treatment, orthognatic surgery, phenotypic expression reflected by TCS severity scores and NOT-S total scores for orofacial dysfunction are presented in Table An experienced otolaryngologist carried out physical examinations of the head and neck, including flexible nasendoscopy. Five participants had been born with cleft palate and all but one child with a submucous cleft had undergone palatal repair. Pharyngeal flap surgery had been performed in one individual early in adulthood. Both children who had been decannulated after long-term tracheostomy were still using manual signing to augment expressive communication.Sixteen of 19 participants were using hearing devices. Six had bilateral bone-anchored hearing aids (BAHA) and two had unilateral. Seven had behind-the-ear aids (BTE), five of which were unilateral and two bilateral. One participant was using bilateral in-the-ear hearing aids (ITE). Pure-tone audiometry (ISO 8253–1 1989) was used to update hearing measures. Pure-tone average (PTA) was calculated for the better ear at frequencies of 500, 1000, 2000 and 4000 Hz . One participant had normal hearing, while four had slight hearing impairment (26–40 dB), seven had moderate (41–60 dB), six had severe (61–80 dB) and one had profound (≥81 dB). The mean PTA dB hearing level was 52.10 (SD 20.04). No differences between age groups or gender were found. Conductive hearing loss was identified in 11 individuals, mixed hearing loss in five and sensorineural loss in two. All except two individuals had appropriately fitted hearing devices. Both were using unilateral hearing aids, but were assessed as requiring bilateral devices.Data on dental occlusion in terms of increased overjet, lateral and anterior open bite and reverse overjet is given in Table TCS severity scores  were calculated by the clinical geneticist according to a system of quantification of the phenotypic expression in TCS developed by Teber et al. . The medThe Nordic Orofacial Test- Screening (NOT-S) was used in assessment of orofacial functions. This protocol contains a structured interview and a clinical examination incorporating six domains with one to five questions or tasks each . The intThirteen sentences from a Norwegian translation of Swedish Articulation and Nasality Test (SVANTE) were used to evaluate articulation, nasality and voice , see AddSimultaneous digital audio recordings  and video recordings  were made using an external condenser microphone . Randomized .wav and .mpeg files were edited in Adobe Premiere 4.0. The sound files were played back using Windows Media Player and the video samples using VLC media player for Windows. Beyerdynamic DT250 headphones were used by the listeners.Production of consonants was first assessed independently by two speech language pathologists in relation to the target articulations . However, the potential challenges in communicative participation in variable social and environmental contexts faced by a broader population have not yet been explored.The low percentage of intelligible words among the youngest participants underlines the need to facilitate speech and communication skills. Considering the high prevalence of deviating speech features and hearing loss, early referral for assessment of speech function and communication is recommended. Early introduction of sign language is often useful to augment communication and language development. One should recognize that the speech composite scores were correlated with the TCS severity scores and the NOT-S scores, which express the close inter-relationship between structural malformations and different orofacial functions . It is iDelayed speech development is one of the items in the severity rating system created by Teber et al. and was reported in a majority of the individuals . UnfortuA comprehensive multidisciplinary protocol outlined by Thompson et al. involving systematic follow-up and treatment planning during maturation has the potential to contribute to improved outcomes in terms of speech and communication, respiration, hearing, and orthodontic status . The preEstablished speech assessment methods have been used to describe speech function. The speech assessment methods have been accurately described in order to ensure reproducibility and make comparisons with larger cohorts possible. Further the speech findings have been related to other features of the same study group to provide better understanding of phenotypic expression of TCS. Interpretation of the findings must be careful due to limited sample size and number of test performed.Unfortunately this study has failed to investigate what factors the children and their parents considered to be limiting in communication. There is clearly a need of a thorough study using an extensive communication questionnaire and in depth interviews to identify the factors that support or undermine communication skills in children and adolescents with TCS. Furthermore, this study only gives an impression of the speech problems at different ages.In conclusion this study showed that children, adolescents and a subgroup of adults had multiple aberrations of articulation, resonance and voice features. Only children presented with markedly reduced intelligibility. Speech dysfunction was significantly correlated with phenotypic severity of TCS. The complexity of speech problems identified in young individuals with TCS indicate that speech monitoring and intervention are required from early in life preferably in multidisciplinary craniofacial team settings. A subgroup of adults with persisting speech deviations may require prolonged attention of speech language pathologists.BAHAm: Bone-anchored hearing aids; BTE: Behind-the-ear hearing aids; CSC: Cleft palate speech characteristics; DHC: Dental Health Component; IPA: International Phonetic Association; ITE: In-the-ear hearing aids; PCC: Percentage correct consonants; NAL: Nasal air leakage; NOT-S: Nordic Orofacial Test- Screening; PTA: Pure-tone average; SVANTE: Swedish Articulation and Nasality Test; TCS: Treacher Collins syndrome; VPC-sum: Velopharyngeal Composite Score- Summary; VPI: Velopharyngeal insufficiency.The authors declare that they have no competing interests.PÅ and CP created the project outline regarding this part of the collaborative project. PÅ acquired the necessary approvals, coordinated the examinations, performed data collection, and wrote the main body of the manuscript draft. CP has taken part in some of the speech analyses and made valuable contributions to the manuscript in her role as supervisor of the master thesis of PÅ. HA contributed with the assessments of hearing and critically revised the manuscript drafts. All collaborators have approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2350/15/47/prepubMaterials for speech assessments.Click here for file