Mycoplasma hyopneumoniae (M. hyopneumoniae). Thirty six-week-old M. hyopneumoniae seronegative piglets were randomly assigned to four groups: three challenged groups with experimentally inoculated pigs by either the endotracheal , intranasal  or aerosol  routes and one uninfected group . Blood samples were collected 1 day before challenge and at necropsy, 28 days post-inoculation (dpi), to assess seroconversion. Laryngeal swabs were collected at −1, 7, 14, 21 and 28 dpi in order to evaluate colonization. At necropsy, lung lesions were scored and lung tissue was collected for histopathological studies and M. hyopneumoniae DNA detection. Broncho-alveolar lavage fluid (BALF) was also obtained to detect M. hyopneumoniae DNA, specific IgA antibodies and cytokines. MP was observed in all inoculated groups, but the ET group displayed a significantly higher number of animals affected by MP as well as a higher mean lung lesion score. These results were paralleled with an earlier seroconversion and upper respiratory tract colonization of M. hyopneumoniae. Additionally, in the ET group, higher levels of pro-inflammatory cytokines and specific IgA antibodies in BALF were found. Under the conditions of the present study, MP was reproduced by the three evaluated inoculation routes. Obtained results suggest that the ET route is the most effective in order to induce MP in pigs experimentally challenged with M. hyopneumoniae.The purpose of this study was to assess the effect of three different inoculation routes into mycoplasmal pneumonia (MP) in pigs challenged with  Mycoplasma hyopneumoniae (M. hyopneumoniae) is the causal agent of mycoplasmal pneumonia (MP). MP is frequently complicated with other bacteria (enzootic pneumonia [EP]) and viruses (porcine respiratory disease complex [PRDC]), which affect the severity of the disease. These chronic respiratory infectious processes affect mainly growing and finishing pigs and are significant causes of economic losses to swine producers throughout the world [he world .M. hyopneumoniae experimental models are essential for vaccine efficacy assessment. However, optimal and reproducible results are not always achieved. In fact, variation in pneumonic lesion severity has been reported in different experimental M. hyopneumoniae challenge systems [M. hyopneumoniae infection pattern and the severity of the associated lung lesions are the strain, duration of the study, type and dose of the inoculum and the inoculation route. The intrinsic virulence of M. hyopneumoniae strains has been demonstrated to determine the clinical course of the infection [M. hyopneumoniae due to the inflammatory response caused by the administration of foreign antigens [ systems –6, also  systems , 6, 7. Snfection . Days lanfection , 9, 10. nfection . With reantigens .M. hyopneumoniae inoculation route used. However, its impact on the pathogenesis of the experimentally induced M. hyopneumoniae infection has been poorly investigated. There are four inoculation routes reported in the peer-reviewed literature used in swine M. hyopneumoniae challenge models: endotracheal (ET), transtracheal (TT), intranasal (IN) and aerosol (AE). Although both intratracheal methods (ET and TT) are the most widely used, MP has been induced by all models. AE is probably the least extended method, despite it is supposed to mimics better the natural conditions of infection [M. hyopneumoniae challenge models using different inoculation routes are scarce. Marois et al. [M. hyopneumoniae, clinical signs and lesion scores were evidenced between infection conditions.One of the most distinguishing features of experimental challenge systems is the nfection . Comparis et al.  comparedM. hyopneumoniae inoculation routes  for their ability to induce MP. The optimum inoculation route was established by studying colonization, clinical, pathological and immunological parameters.This last point should be subjected to further investigation. Therefore, the aim of this study was to compare three M. hyopneumoniae and Porcine reproductive and respiratory syndrome virus (PRRSV) based on serology and clinical history. For animal selection, serology  and a nested PCR (nPCR) for detection of M. hyopneumoniae DNA [®) into four groups equalled according to body weight. Challenged animals were comingled in the same room whereas the control group was placed in a separated room.Animals were obtained from a herd located in North-Eastern Spain that was free from niae DNA  was donen = 24) were inoculated with 5 mL of M. hyopneumoniae fresh culture on two consecutive days. Two control animals received 5 mL of sterile phosphate buffered saline (PBS) on two consecutive days by one of the three assessed routes . At 28 days post-inoculation (dpi), all pigs were euthanized with an intravenous overdose of sodium pentobarbital and subjected to necropsy examination.At approximately 6 weeks of age, pigs were challenged according to the experimental design detailed in Table Study procedure was approved by the Animal Experimentation Ethics Committee of the Universitat Autònoma de Barcelona (n°= 5796) and of A.M. Animalia Bianya S.L. (n°= 05/15).M. hyopneumoniae field strain was used as the inoculum. This strain was isolated in 2010 from a lung of a slaughter-age animal showing MP. Inoculum titre was determined by using a limiting dilution method. Briefly, ten-fold dilutions of the inoculum were made and left to grow for 2 weeks at 37 °C. Tubes were tested for M. hyopneumoniae by PCR [50/mL, in which PCR50 represents the limiting dilution of the inoculum that is PCR positive in 50% of its replicates.A fresh culture derived from a e by PCR  at 1 ande by PCR . The ino®; Merial, France), 0.4 mg/kg Butorfanol  and 6 mg/kg of Azaperone  and placed in sternal recumbence. The inoculum was administered through an individual mask  connected to an aerosol delivery system  with a total output rate of approximately 600 mg/min and a particle mass median diameter of 3.5 µm under a pressure of 1.6 bar.For the ET inoculation, a double catheter   was introduced in the trachea. The inoculum was administered with a syringe through the internal catheter. For the IN inoculation, a mucosal atomization device  attached to a syringe was used to administrate half of the inoculum volume into each nostril. Animals included in the AE group, were anaesthetised with a combination of 10 mg/kg Ketamine  was calculated according to the following formula: body weight at necropsy minus the body weight before challenge divided by the days lapsed between them. g for 10 min at 4 °C and sera were stored at −80 °C until used. Laryngeal swabs were resuspended in 1 mL sterile PBS, vortexed and stored at −80 °C.Blood was collected 1 day before challenge (−1 dpi) and at necropsy day (28 dpi). Laryngeal swabs were obtained as described previously  at −1 dpM. hyopneumoniae DNA respectively. Broncho–alveolar lavage fluid (BALF) was collected from twelve animals, selecting those three animals showing the most severe lung lesions (when present) within each group. BALF was left for gross mucus sedimentation and supernatant was stored at −80 °C until it was processed. These samples were used for M. hyopneumoniae DNA, cytokine and specific IgA antibody detection.At necropsy (28 dpi), two lung samples were collected from all each animals: one was fixed in 10% neutral buffered formalin and the second one was frozen. These samples were used for histopathological studies and detection of M. hyopneumoniae infection  was assessed using the European Pharmacopoeia  scoring system. For histopathological studies, formalin-fixed tissues were processed routinely and embedded in paraffin wax. Sections (4 μm) were stained with haematoxylin and eosin and examined under light microscope for broncho-interstitial pneumonia (BIP). Microscopic scoring was performed as previously described [The extension of gross lung lesions compatible with escribed . BrieflyM. hyopneumoniae antibodies by means of a commercial competitive inhibition enzyme-linked immunosorbent assay . Samples with mean optical density (OD) <50% of the OD of the buffer control were considered positive. Doubtful (OD from 50 to 64%) and negative (OD ≥ 65%) OD-values were classified as negative in the statistical analysis.Sera were tested in duplicate for M. hyopneumoniae specific IgA in BALF was performed modifying the Mycoplasma hyopneumoniae Antibody Test Kit  with an alkaline phosphatase-labelled goat anti-porcine IgA polyclonal antibody  at 1:5000 dilution as secondary antibody. BALF samples were tested undiluted and in duplicate. One hundred µL of each sample were used in the ELISA assay. The cut-off was established at mean OD value of BALF from the control animals plus three folds the standard deviation (SD). Values higher than the cut-off were considered positive. Values below this cut-off were considered negative.Detection of ® 96 DNA Blood kit  on the BioSprint 96 workstation . Lung tissue was disrupted using TissueLyser  for DNA extraction. Approximately 1 g of tissue was homogenized with 600 µL of PBS into plastic tubes containing glass beads. After shaking for 10 min, the lung homogenate was centrifuged at 11 000 g for 1 min. DNA was extracted from 200 μL of tissue supernatant  according to the manufacturer’s instructions on the BioSprint 96 workstation . To assess potential contamination during the extraction procedure, a negative control was included using PBS as an extraction substrate in each extraction plate.DNA was extracted from 200 μL of laryngeal swabs suspension or undiluted BALF using BioSprintM. hyopneumoniae PCR (rt-PCR) was performed in laryngeal swabs, BALF and lung tissue samples. The assay was performed using VetMAX™-Plus qPCR Master Mix  and VetMAX™ M. hyopneumoniae Reagents , according to the manufacturer’s instructions. VetMAX™-Plus qPCR Master Mix kit includes Xeno™ DNA Control, which serves as an internal positive control for DNA purification and rt-PCR. Runs were carried out in an ABIPRISM® 7500 machine . The threshold for the DNA target was set at 10% of the average maximum fluorescence value of the positive control DNA target. Cycle threshold (Ct) values equal to or lower than 40 were considered positive.A commercial real time ® ELISA kits  following manufacturer’s recommendations. BALF samples were tested undiluted. Reactions were measured using OD at 450 nm and quantified by the use of a standard curve.Levels of pro-inflammatory cytokines IL-1β, IL-8, IL-6 and TNF-α were determined using a commercially available Porcine QuantikineP values ≤ 0.05 were considered statistically significant, whereas p values > 0.05 and ≤ 0.10 were considered statistical tendencies.Statistical analyses were performed using NCSS software . Normal M. hyopneumoniae infection throughout the study.Negative control pigs were negative for antibodies, clinical signs, DNA and lesions associated to Very sporadic and mild coughing was displayed by challenged pigs from 14 dpi onwards. No other significant clinical signs were observed during the experiment. No significant differences in mean body weight  neither in ADWG were observed among groups (data not shown).M. hyopneumoniae DNA by rt-PCR in lung tissue.MP was recognised taking into account three criteria concomitantly: (1) Presence of CVPC, based on gross evaluation, (2) presence of BIP determined through microscopic evaluation (score 3 or 4) and (3) detection of p < 0.05).Percentage of animals showing MP and the mean lung score within each group are represented in Table p < 0.05). The ET mean percentage of inhibition was also statically lower (p < 0.01) than all the other groups except for the IN. Interestingly, all animals that seroconverted showed MP regardless the experimental group.None of the animals was seropositive 1 day before challenge  mean OD value than those of the IN, AE and control groups .The cut-off value of the indirect IgA ELISA was established at an OD value of 0.21. All BALF samples belonging to the challenged groups  were M. hyopneumoniae rt-PCR and their Ct values (mean ± SD) in laryngeal swabs along the study, in lung tissue and BALF within each group are represented in Table The percentage of positive animals to M. hyopneumoniae DNA was detected from 7 dpi onwards, in both IN and AE groups, M. hyopneumoniae DNA was not detected until 21 dpi. Independently of the group, all animals that became positive in a certain time point of the study remained positive until the end (28 dpi). The highest percentage of positive animals was detected at 21 dpi in all groups but except for the IN group, in which rt-PCR positive animals appeared to increase over the last week (28 dpi). In fact, the maximum number of positive animals (75%) within a group was present in the IN group at 28 dpi. All the animals showing MP were rt-PCR positive in laryngeal swabs at necropsy day, except two animals belonging to the ET group. No significant differences in mean Ct values were observed between groups at any time point.All the animals included in the study were negative to rt-PCR in laryngeal swabs before challenge. While in the ET group M. hyopneumoniae rt-PCR positive animals in lung tissue were detected in all challenged groups, but the highest percentage of positive animals belonged to the ET one. All animals with MP were rt-PCR positive in lung tissue at necropsy day regardless of the group. However, three animals without MP from the IN group were also lung rt-PCR positive. From the total of the 15 animals that were M. hyopneumoniae positive in lung tissue, 11 were also positive in laryngeal swabs at the same time point of the study (28 dpi), showing both techniques a coincidence of 77% (κ = 0.53). Although in the IN group 5 out of 8 animals (62.5%) were rt-PCR positive, mean lung tissue Ct value was significantly (p < 0.05) higher in this group compared to the other two challenged groups.All the BALF samples from the three challenged groups  were rt-PCR positive. No significant differences in mean Ct values were observed in BALF between groups.p ≤ 0.10). Remarkably, mean levels of IL-1β (p < 0.001) increased significantly in ET-challenged pigs in comparison with mean levels found in all other groups . Mean TNF-α Figure A and IL-s Figure C.Figure M. hyopneumoniae using three different inoculation routes was assessed in the present study. Lung lesion scores have been previously reported to be similar between groups of pigs experimentally challenged by means of different inoculation routes [M. hyopneumoniae infectious doses and necropsy timings were used, difficulting comparisons with the present study, as such factors are known to influence the infection pattern and MP severity in pigs [M. hyopneumoniae.The ability to induce MP in 6-week-old conventional piglets experimentally challenged with n routes . However in pigs . To the The three tested inoculation routes  had been already reported to induce MP , 12, 19.M. hyopneumoniae is primarily found on the mucosal surface of the trachea, bronchi and bronchioli [M. hyopneumoniae in infected live pigs using nested or real-time PCR assays [M. hyopneumoniae infection has been recently reported [M. hyopneumoniae detection by rt-PCR was found at necropsy. In the present report, colonization timing differences were found between groups; M. hyopneumoniae DNA was detected in the ET group 2 weeks earlier (7 dpi) than in the IN and AE groups (21 dpi). Although only two out of eight (25%) IN-challenged animals were showing MP, laryngeal swabs rt-PCR positive animals appeared to increase over the last week (28 dpi) in this group, accompanied by quite a high proportion (5 out of 8) of rt-PCR positive animals in lung tissue at that time. It has been reported that M. hyopneumoniae gross lung lesions reached their maximum score by 28 dpi [onchioli , and itsonchioli . PreviouR assays , 22, 23.reported . Under tf eight 2% IN-chaly 28 dpi , 10. In M. hyopneumoniae shedding cannot be completely excluded. However, this event would be very unlikely since the first evidence of M. hyopneumoniae infection was seen at 7 dpi on two animals from the ET group. Considering that necropsies were done 3 weeks after this first detection, the probability that seroconversion and lung lesions observed in AE and IN-challenged animals were due to these two pigs is probably very low.One limitation of this study was the fact that challenged animals were comingled in the same room and transmission between groups was possible throughout the study. In consequence, whether the infection observed in IN and AE groups is product of the ET M. hyopneumoniae, which then is reflected on its colonization and in consequence, on the infection outcome. Information regarding how the inoculation route can affect the survival and infectivity of M. hyopneumoniae particles is limited. By means of the ET route, the inoculum penetration into the lower respiratory tract is expected to be higher, which might enhance colonization. Although IN and, more importantly, the AE inoculation routes mimic better the natural infection conditions, the pig’s long and curving respiratory tract may represent a major barrier for such administrations [M. hyopneumoniae bacterium mean diameter is 0.20 µm [M. hyopneumoniae deposition in the respiratory tract.Among many other factors, development of MP is dependent on the number of organisms that colonize the respiratory tract, which is likely dependent on cumulated infectious doses . Since atrations . Relatedtrations . While M 0.20 µm , deposit 0.20 µm , 27, a rM. hyopneumoniae and, therefore, a higher exposure to the mucosal immune system. In fact, all animals that seroconverted at necropsy had also MP. Indeed, it is well known that the concentration of serum antibodies does not correlate with clinical protection against M. hyopneumoniae [M. hyopneumoniae challenge in BALF did not prevent MP development. Since the three BALF samples that were taken per group belonged to those animals with the highest lung scores (when MP was present) and comparisons with those animals without lesions within a group could not be performed, BALF results are likely overestimated.In the current study, a significantly earlier seroconversion and lower percentages of inhibition were found in the ET group. The reason for this finding might be due to the higher cumulated infectious dose achieved via the ET inoculation route, which might imply a faster colonization of eumoniae , 29. Neveumoniae –33, the eumoniae , where IM. hyopneumoniae infected pigs [The evaluation of the mucosal immune response was further addressed by the analysis of cytokine levels in BALF. Cytokine responses in BALF from control animals were found to be low and within a clinically normal range by comparing with previously reported data , 10, 32.ted pigs , 35 and ted pigs  have alrted pigs , 10, 34.M. hyopneumoniae colonization and immune response against infection, which at the end is reflected in a higher incidence and more severe MP.Under the conditions of this study, ET inoculation route was more effective inducing MP 4 weeks after challenge than the IN or AE ones. ET route is expected to apply a greater inoculum volume in pig’s lower respiratory tract, achieving greater infectious doses in shorter times and promoting an earlier


Postoperative hearing loss following nonotologic surgery is rare. For patients undergoing subarachnoid anesthesia, the loss of cerebral spinal fluid and hence the drop in intracranial pressure can result in hearing loss and cranial nerve palsy. We report a case in which a patient sustained orthopedic surgery under combined spinal and epidural anesthesia complicated by severe and persistent sensorineural hearing loss. This report is a reminder that postoperative sudden sensorineural hearing loss is a poorly understood complication. A high index of suspicion is required for early diagnosis of this complication, although prompt treatment does not guarantee a good outcome. Postoperative sensorineural hearing loss (SNHL) following nonotologic surgery is rare and often detected only by audiometric evaluation –3. This μg fentanyl was injected into his subarachnoid space. CSEA has been uneventfully performed using needle through needle technique in the sitting position at L3-4 intervertebral space by midline approach at the first attempt. During surgery, an additional 500 mL of lactated Ringer's solution was administered. Surgery was completed within 100 minutes and was otherwise uneventful, and hemodynamic parameters remained within normal limits throughout the perioperative period. A unilateral left-sided SNHL was discovered shortly after completion of surgery in the recovery. This was associated with mild left-side tinnitus but no vertigo. There was no aural fullness, pain, headache, or postural element to his symptoms. Otoscopic examination was normal, Weber and Rinne tests suggested left-sided sensorineural hearing loss. A subsequent pure tone audiogram showed a bilateral SNHL, severe on the left side and moderate on the right side, and speech discrimination scores were 60% on the left and 80% on the right side , an 18-gauge (G) epidural needle was inserted and loss of resistance technique was used to identify the epidural space followed by insertion of the 27 G pencil point spinal needle. When spontaneous backflow of CSF was observed, intrathecal 12.5 mg of heavy bupivacaine with 15 ght side . Additioght side .Tinnitus combined with SNHL following either general or spinal anesthesia has been documented –3. HeariNo substantial change in hearing levels was observed with the initial management . After initiation of hyperbaric oxygen therapy, hearing improvement was noted. At the end of the treatment, the patient's hearing loss improved from severe to moderate level and became almost equivalent to the contralateral side.To the best of our knowledge, there are no prior accounts in the literature on the successful use of hyperbaric oxygen therapy in the treatment of SNHL following spinal anesthesia.This report serves to remind clinicians that postoperative sudden SNHL is a poorly understood complication. A high index of suspicion is required for early diagnosis of this complication, although prompt treatment does not guarantee a good outcome. Since it is not possible at this time to anticipate which patients are at highest risk of developing SNHL after spinal anesthesia, prompt attention should be given to any patient complaining of hearing loss or tinnitus.


Amblyomma variegatum. This peptide, named amregulin, is composed of 40 residues with an amino acid sequence of HLHMHGNGATQVFKPRLVLKCPNAAQLIQPGKLQRQLLLQ. A cDNA of the precursor peptide was obtained from the National Center for Biotechnology Information . In rat splenocytes, amregulin exerts significant anti-inflammatory effects by inhibiting the secretion of inflammatory factors in vitro, such as tumor necrosis factor-alpha (TNF-α), interleukin-1 (IL-1), interleukin-8 (IL-8) and interferon-gamma (IFN-γ). In rat splenocytes, treated with amregulin, compared to lipopolysaccharide (LPS) alone, the inhibition of the above inflammatory factors was significant at all tested concentrations . Amregulin shows strong free radical scavenging and antioxidant activities  in vitro. Amregulin also significantly inhibits adjuvant-induced paw inflammation in mouse models in vivo. This peptide may facilitate the ticks’ successful blood feeding and may lead to host immunotolerance of the tick. These findings have important implications for the understanding of tick-host interactions and the co-evolution between ticks and the viruses that they bear.Ixodid ticks are well known for spreading transmitted tick-borne pathogens while being attached to their hosts for almost 1–2 weeks to obtain blood meals. Thus, they must secrete many immunosuppressant factors to combat the hosts’ immune system. In the present work, we investigated an immunosuppressant peptide of the hard tick  Ixodid ticks are important arthropod vectors that transmit pathogens to other animals. The saliva of these ticks contains a cocktail of dozens of different bioactive molecules, including coagulation inhibitors, immunomodulatory molecules and, in many ixodid genera, toxins, which may cause acute, progressive and potentially fatal muscle paralysis . It has A. variegatum has been identified as a vector for pathogens, such as Rickettsia spp.  [Ehrlichia ruminantium (heart water disease) [A. variegatum have also recently been identified. Because most of these proteins have no known function, functional analysis of these proteins with the aim of discovering novel pharmacologically-active compounds is of considerable interest.Medically-significant tick-borne diseases include Lyme disease ,9, babesections)  and Ehrldisease) . Novel sBorrelia burgdorferi spp., the causative agent of Lyme disease [A. variegatum and named amregulin was obtained from NCBI. This peptide suppresses the host inflammatory response by inhibiting cytokine secretion and detoxifying reactive oxygen species. Furthermore, the reliability of amregulin's immunosuppression function in vivo was also examined in this work.Several studies have reported that secretions (mainly proteins) extracted from tick saliva and salivary glands may inhibit host humoral immunity, as well as B- and T-cell responses to tick-transmitted pathogens ,20,21. M disease ,30. It h disease ,20,31,32The cDNA of amregulin encodes a precursor protein composed of 59 amino acid (aa) residues with the sequence of MKLHMLNMLNCLLLTVCDGHLHMHGNGATQVFKPRLVLKCPNAAQLIQPGKLQRQLLLQ. The mature peptide is composed of 40 residues with the sequence of HLHMHGNGATQVFKPRLVLKCPNAAQLIQPGKLQRQLLLQ .vs. 3720 ± 25.3 pg/mL). TNF-α, IL-1 and IL-8 were also significantly inhibited by amregulin, as compared to the LPS-treated control groups. A scrambled control peptide was also used as a control. At every test concentration, the control showed no effects on the secreted amounts of the different cytokines. These results collectively indicate that amregulin markedly inhibits the LPS-induced secretions of four different inflammatory cytokines in rat splenocytes in a concentration-dependent manner.To evaluate the immunosuppression capabilities of amregulin, its effects on IL-1, IL-8, IFN-γ and TNF-α secretions induced by LPS in rat splenocytes were tested. Four different concentrations  of amregulin were used to stimulate rat splenocytes, either alone or in concert with LPS. Treatment with LPS alone strongly induced the secretion of IL-1, IL-8, IFN-γ and TNF-α, whereas amregulin markedly inhibited these LPS-induced cytokine secretions in a dose-dependent manner A–D. The +) radicals are commonly used to evaluate the antioxidant capacity of biomolecules due to their maneuverability, relative stability and good reproducibility [+ by measuring decreases in absorbance at 517 and 734 nm, respectively. Amregulin showed a strong, concentration-dependent scavenging ability toward ABTS+, as compared to both a negative (scrambled control peptide) control and a positive butylated hydroxytoluene (BHT) control, but not toward DPPH, as compared to a BHT positive control. The scrambled control peptide showed very limited free radical scavenging activity (even at a concentration of 20 µg/mL). Nevertheless, amregulin’s ability to scavenge DPPH free radicals also became stronger with increasing concentrations. At concentrations of 10 and 20 µg/mL, amregulin also showed a strong, concentration-dependent ability to quench Nitric Monoxide (NO), which corresponds to its ability to reduce DPPH and ABTS+ free radicals. These results indicate that amregulin possesses strong free radical scavenging capacity  and 2,2′-azinobis 3-ethylbenzothiazoline-6-sulfonic acid (ABTScibility . Reactiocapacity .3+) by a Fe3+–Fe2+ transformation method. As shown in We determined the capacity of amregulin to reduce ferric ions  to evaluate the potential anti-inflammatory ability of amregulin in vivo. As indicated in the Experimental Section, the basal footpad thickness of different male Kunming mice after treatment was selected as an indicator of inflammation. Along with the increments in treatment time, the anti-inflammatory effects of amregulin were enhanced with increasing concentrations, whereas the scrambled control peptide (8 mg/kg bodyweight) showed nearly no effects on inflammation. Significant anti-inflammatory effects were present in all mice treated with different concentrations  of amregulin; the paw inflammation was significantly decreased compared to that of the negative controls. The mice treated with 4 and 8 mg/kg body weight recovered to normal status after 14 days of administration. These results collectively indicate that amregulin inhibits hind paw inflammation in mice in a concentration-dependent manner  or many (I. uriae) such viruses [The progress of tick feeding not only causes direct injury to the host’s skin, but also provides opportunities for disease-causing organisms to be ingested or expelled by the parasite. Thus, the host inflammatory and immune responses must be initiated. IL-1 was first discovered because of its strong pro-inflammatory effects. It induces a complex network of pro-inflammatory cytokines that initiate and regulate inflammatory responses via the expression of various integrins on leukocytes and endothelial cells. IL-8 induces chemotaxis of target cells, primarily neutrophils, as well as other granulocytes, causing them to migrate toward the site of infection. In humans, IL-8 is believed to play an important role in the pathogenesis of bronchiolitis , which i viruses . Determi viruses . The eff3+ iron, indicating that amregulin may be an important peptide in the blood feeding process of A. variegatum.Beyond its strong antioxidant activity, amregulin was found to scavenge free radicals in a concentration-dependent manner. It has been demonstrated that ABTS radicals react with phenols to form purple products with a broad absorbance of approximately 550 nm ,42. Of tin vivo research by using an adjuvant-induced inflamed mouse paw model to demonstrate the potential of amregulin to suppress inflammation and noted concentration-dependent inhibition of inflammation. At Day 11, the group treated with 8 mg/kg amregulin differed significantly from the negative control group. At Day 14, the same group showed no difference in paw thickness from normal mice. The 4 mg/kg amregulin-treated group showed greatly decreased paw inflammation, whereas the scrambled control peptide showed virtually no effects on inflammation. Each amregulin-treated group showed that the administration of this peptide was able to substantially control the development of inflammation. This result demonstrates the biological significance of this immunosuppressant peptide derived from hard tick salivary glands and contributes to further understanding of the mechanism underlying hard tick immunosuppressive properties against host inflammatory responses.Beyond showing the antioxidant and anti-inflammatory effects of amregulin at molecular and cellular levels, we also performed Although the synthetic peptide may not be the same as the endogenous form because of posttranslational modifications, such as glycosylation or interactions with other proteins, or may even form a dimer via the cys residue, our findings may still provide some implications for understanding the mechanisms of successful tick hematophagy and tick-host interactions.in vivo. Together, the results of this study suggest that the biological activities of amregulin facilitate successful parasite feeding.The present work focused on the peptide amregulin, which exerts significant anti-inflammatory effects by inhibiting host secretion of the inflammatory factors TNF-α, IL-1, IL-8 and IFN-γ. It also shows strong antioxidant properties. Amregulin effectively inhibited the adjuvant-induced inflammation of mouse paws A. variegatum. The protein precursor encoded by the cDNA was analyzed by SignalP 4.0  encoding the precursor of amregulin was obtained from the National Center for Biotechnology Information  . The cDNrk, 2016 ) to pred6 cells (100 µL per well) were incubated at 37 °C and 3.5% CO2. Different concentrations of the peptide were prepared by taking 20-µL samples of previously prepared peptide stock solution and dissolving them in different volumes of RPMI 1640 medium with LPS . These different peptide solutions were then co-cultured with splenocytes. After a 48-h cultivation, culture supernatants were harvested and stored at −70 °C. All combinations were replicated three times. Cytokine assays for IL-1, IFN-γ, IL-8 and TNF-α were conducted by using antibody-sandwich ELISAs  according to the manufacturer’s instructions.The wells of a 96-well plate were seeded with a suspension of splenocytes from Wistar rats in RPMI 1640 medium  supplemented with 5% fetal bovine serum and 100 U/mL penicillin and streptomycin. A total of 6 × 105 M DPPH radical dissolved in ethanol and 0.1 mL of sample solution . Following 30 min of incubation at room temperature, absorbance was red against a blank at 517 nm. The percentage inhibition of DPPH free radicals by the peptide (I%) was calculated according to Equation (1):Ablank is the absorbance of the control reaction (reaction mixture minus the test compound) and Asample is the absorbance of the test compound. Butylated hydroxytoluene  and a scrambled control peptide were used as a positive and a negative control, respectively.We determined free radical scavenging activity by using a stable DPPH radical (Sigma), slightly modifying a previously-reported method ,42. The + with a GMS10114.4 kit  according to the manufacturer’s instructions, using BHT as a positive control and the scrambled control peptide as a negative control. We used a linear regression analysis to determine the total formation of products  and the total consumption of the ABTS radical. The concentrations of ABTS+ and ABTS were calculated to be ε340 = 4.8 × 104 M−1·cm−1 and ε415 = 3.6 × 104 M−1·cm−1, respectively. The purple end product of ABTS+ with amregulin was monitored by measuring absorbance at 550 nm.We further determined free radical scavenging activity by measuring the reduction of ABTSWe determined the NO scavenging capacity of amregulin by incubating 5 mM sodium nitroprusside dihydrate  in phosphate-buffered saline  with different concentrations of peptide samples at 25 °C, and 0.5 mL of the incubation solution was withdrawn and mixed with 0.5 mL of Griess reagent  after 20 min. Absorbance was measured at 550 nm. The amount of nitrite was calculated from a standard curve constructed using sodium nitrite.3+ was evaluated using the method developed by Wu et al. [3Fe(CN)6. The mixture was incubated at 50 °C for 20 min, after which 2.5 mL of 10% trichloroacetic acid (TCA) was added to the mixture, and this was followed by centrifugation at 3000 rpm for 10 min. Of the solution, 2.5 mL of the upper layer was mixed with an equal volume of distilled water and 0.5 mL of FeCl3 (0.1%), and the absorbance was measured at 700 nm. An increase in the absorbance of the reaction mixture indicated reduction of Fe3+. BHT was again used as a positive control.The reducing power of amregulin against Feu et al. . VaryingTwenty microliters of vehicle  or Freund’s complete adjuvant (Sigma) was administered to the plantar surface of the right hind paws of male Kunming mice (12/group). The basal footpad thickness of each mouse was measured with a vernier caliper at the beginning of the experiment. Amregulin  was administered to the rear femoral muscle on alternating days from Day 2 after injection of Freund's complete adjuvant  to Day 14. The control groups of mice received the same volume of saline (vehicle). Right hind paw thickness was used as an indicator of inflammation and measured with a vernier caliper on Days 0, 1, 4, 8, 11, and 14. The experimental protocols were approved by the Animal Care and Use Committee at Nanjing Agricultural University .t-test. p-values less than 0.05 were considered to be statistically significant, whereas p-values less than 0.01 were considered extremely statistically significant.All data are presented as means ± SD and were analyzed by a one-way or two-way analysis of variance (ANOVA), followed by Duncan’s test or an unpaired in vivo experiments were performed according to sound practices of laboratory animal management.The Animal Care and Use Committee of Nanjing Agricultural University evaluated and approved all procedures used in this study. All


Horizontal gene transfer (HGT) drives the evolution of recipient organism particularly if it provides a novel function which enhances the fitness or its adaption to the environment. Virus-host co-evolution is attractive for studying co-evolutionary processes, since viruses strictly replicate inside of the host cells and thus their evolution is inexorably tangled with host biology. HGT, as a mechanism of co-evolution between human and viruses, has been widely documented, however, the roles HGT play during the interaction between human and viruses are still in their infancy. In this study, we performed a comprehensive analysis on the genes horizontally transferred between viruses and their corresponding human hosts. Our study suggests that the HGT genes in human are predominantly enriched in immune related GO terms while viral HGT genes are tend to be encoded by viruses which promote the invasion of immune system of hosts. Based on our results, it gives us a hint about the evolution trajectory of HGT events. Overall, our study suggests that the HGT between human and viruses are highly relevant to immune interaction and probably reshaped the arm race between hosts and viruses. After classification of those HGT genes, we performed a GO enrichment analysis. In total 46 GO terms were found to be enriched with the p value smaller than 0.05  receptor(CCR) 1–7, CCR9, CCR-like 1 and CCR-like 2, C-X-C motif chemokine receptor 3, 4 and 6) harbor the best hit with the same viral protein, G-protein coupled receptor E1 (Q89609) encoded by equine herpesvirus 2 (EHV-2). The identity between those 13 host proteins and E1 vary from 33.5% to 55.8%. As the identity between CCR3 and E1 is the highest (55.8%), we performed a comparison analysis of protein sequence, transmembrane domains and 3D structure between CCR3 and E1. Overall, CCR3 and E1 exhibit very high degree of similarity. Due to alternative splicing, CCR3 possesses 6 isoforms CCR3-001 , CCR3-201, CCR3-002 , CCR3-007 , CCR3-003  and CCR3-004 . E1 is composed of 1149 nucleotides, which is subsequently translated into 383 aa, with 28 more aa than CCR3-001. TMHMMGene duplication serves as an important source of functional divergence, as duplicated genes may gain novel functions or lose original ones through modifying their existing regulatory network by accumulation of mutations in coding or non-coding regions4042The gene flow between viruses and hosts normally happens after the integration of viral genomic fragments or whole genomes into host chromosomes. This phenomenon occurs frequently in retroviruses such as human immunodeficiency virus (HIV) through the integration of double stranded DNA (dsDNA) which were reverse transcripted from viral RNA genome44464749et al., the direction of gene transfer between viruses and humans remains an open question515354555657585961In a recent report by Crisp The comparison analysis suggests a high similarity of both secondary structure and 3D structure between viral protein E1 and host protein CCR3, implying they share similar biological functions as well. As CCR3 is well known for its importance in inducing the migration of immune cells into inflammatory sites6364The genes lists were analysed using PANTHERThe virus protein database were created using makeblast command from blast package, using the virus sequences downloaded from UniprotThe transmembrane protein topology of E1 and CCR3 were predicted with TMHMMThe orthlogs of human CCRs were downloaded from KEGG database and analysed using DIVERGE 342How to cite this article: Chen, D.S. et al. Horizontal gene transfer events reshape the global landscape of arm race between viruses and homo sapiens. Sci. Rep.6, 26934; doi: 10.1038/srep26934 (2016).


Reduced hearing acuity is among the most prevalent of chronic medical conditions among older adults. An experiment is reported in which comprehension of spoken sentences was tested for older adults with good hearing acuity or with a mild-to-moderate hearing loss, and young adults with age-normal hearing. Comprehension was measured by participants’ ability to determine the agent of an action in sentences that expressed this relation with a syntactically less complex subject-relative construction or a syntactically more complex object-relative construction. Agency determination was further challenged by inserting a prepositional phrase into sentences between the person performing an action and the action being performed. As a control, prepositional phrases of equivalent length were also inserted into sentences in a non-disruptive position. Effects on sentence comprehension of age, hearing acuity, prepositional phrase placement and sound level of stimulus presentations appeared only for comprehension of sentences with the more syntactically complex object-relative structures. Working memory as tested by reading span scores accounted for a significant amount of the variance in comprehension accuracy. Once working memory capacity and hearing acuity were taken into account, chronological age among the older adults contributed no further variance to comprehension accuracy. Results are discussed in terms of the positive and negative effects of sensory–cognitive interactions in comprehension of spoken sentences and lend support to a framework in which domain-general executive resources, notably verbal working memory, play a role in both linguistic and perceptual processing. Unlike reading, where one can control the input rate with eye-movements, in the case of spoken language speech rate is controlled by the speaker and not by the listener. Because of the rapidity of natural speech and its inherently transient nature, comprehension operations that cannot be accomplished as the speech is being heard must be conducted on a fading trace of that speech in memory . Added tAdult aging brings special challenges for speech comprehension due to age-related declines in episodic memory , processAge-related hearing loss is the third most prevalent chronic medical condition among older adults, exceeded only by arthritis and hypertension . This isWhen the consequences of this front-end perceptual effort are added to an age-related decline in working memory capacity e.g., , one migThis delicate balance between the negative effects of processing deficits and the positive effects of spared linguistic knowledge in adult aging works well until the total processing burden exceeds a listener’s processing capacity. When compensatory mechanisms are not able to keep up with demand, listeners’ performance will suffer. Increasing the processing challenge through linguistic and acoustic manipulations is therefore a useful way to test the interaction of cognitive and perceptual factors in speech comprehension. In the following experiment, we examine spoken sentence comprehension under conditions in which this balance is maintained, and under conditions where the processing challenge disrupts this balance by increasing the processing demands needed for successful comprehension at the linguistic and perceptual levels.agent] assisted [action] the queen [recipient of the action]”). When sentences become longer, or the sentence meaning is represented with complex syntax, the cognitive challenge becomes greater : a six-word sentence with a subject-relative center-embedded clause structure, in which the main clause (Sisters are fortunate) is interrupted by a relative clause (that assist brothers). The more complex syntactic form we employed had an object-relative center-embedded clause structure. The first sentence in the lower set shows a sentence composed of the same six words, but now ordered such that the meaning is expressed with an object-relative construction. In this case the embedded clause (that sisters assist) not only interrupts the main clause, but the head noun phrase (Brothers) functions as both the subject of the main clause (brothers are fortunate) and the object of the relative clause (that sisters assist). Because the order of thematic roles in object-relative constructions is not canonical (the first noun is not the agent of the action), such sentences require a more extensive thematic integration than required for the more canonical structure represented by subject-relative sentences  into each of the six-word base sentences. Moreover, the particular placement of the prepositional phrase manipulated the processing challenge by manipulating the separation between key sentence constituents. In a less syntactically disrupting case the placement of the prepositional phrase kept the person performing the action and the action being performed adjacent to, or in close proximity with, each other. These are indicated in Table 1 as short separation sentences. The second sentence in the upper set illustrates such a propositional phrase placement for a subject-relative sentence (short separation). The second sentence in the lower set shows this for an object relative sentence. To test these hypotheses, 10-word sentences were created from the six-word base sentences by inserting a four-word prepositional phrase  and an object-relative sentence (lower set). If object-relative sentences prove more difficult, this manipulation would allow us to dissociate the challenging grammatical features of this sentence from the increased difficulty associated with the increased separation between the key sentence constituents.In the second type of placement the prepositional phrase was inserted in a position to produce a long separation between the person performing the action and the action being performed. This placement was designed to add difficulty to the task of determining the thematic role assignments of the two persons in the sentence, and a presumed increase in working memory demands, but without changing the formal syntactic structure of the sentence itself. Examples of such that was used instead of the more grammatically correct who. This was done to avoid the use of the who–whom distinction that could serve as an undesired comprehension cue.)By having sentences in which males  or females  as the agents or recipients of actions, accurate comprehension could be demonstrated by the participant correctly indicating the gender of the agent of the action.  or at a presentation level relative to an individual’s hearing threshold . Experimental studies typically employ either one presentation method or the other; rarely both within the same experiment. This leaves open the question of whether the two methods will be equally sensitive to the factors of interest in a particular study. For this reason in the present study, we employed both of the presentation methods  using a within-participants design. Including both sound presentation levels would thus allow us to see whether both methods may reveal an influence on the factors tested in this sentence processing task equally, and to provide useful empirical information in helping to determine which approach may be more appropriate in future studies. Thus, uniquely within a single experiment, we manipulate syntactic complexity, the effect of a separation of key sentence elements by insertion of a prepositional phrase, and presentation level of the sentence stimuli within the context of adult aging and hearing acuity.One could entertain two hypotheses in terms of sentence comprehension in older adults with good or poor hearing acuity. The first is that perceptual effort – as determined by participants’ hearing acuity and presentation level – will have similar effects on sentence comprehension regardless of the cognitive load imposed by syntactic complexity, sentence length, and prepositional phrase placement. This simple additivity would be manifested in parallel comprehension performance functions for the good-hearing and hearing-impaired listeners, albeit with a potential difference in y-intercepts. A finding of additivity would be consistent with the notion of independence of cognitive and perceptual operations.  than on comprehension of the less challenging sentences (subject-relative sentences with a short agent-action separation).This latter finding would be in keeping with the principles embodied in models that postulate limited attentional  or workiParticipants were 36 older adults, 18 with good hearing acuity  and 18 older adults with a mild-to-moderate hearing loss . Audiometric assessment was conducted using a GSI 61 clinical audiometer  using standard audiometric procedures in a sound attenuating testing room.Figure 1 shows better-ear pure-tone thresholds from 500 to 8,000 Hz for the three participant groups plotted in the form of audiograms, with the x-axis showing the test frequencies and the y-axis showing the minimum sound level (dB HL) needed for their detection. Hearing profiles for individual listeners within each participant group are shown in light gray, with the group average drawn in black. The shaded area in each of the panels indicates thresholds less than 25 dB HL, a region commonly considered as clinically normal hearing for speech (r speech .SD = 3.0). The older adult hearing-impaired group had a mean PTA of 35.8 dB HL (SD = 5.7) placing them in the mild-to-moderate hearing loss range  across.5, 1, 2, and 4 kHz, a range especially important for the perception of speech. The participants in the older adult good-hearing group had a mean PTA of 17.9 dB HL  and the hearing-impaired group ranging from 67 to 83 years . Both groups were well educated, with a mean of 16.5 years of formal education for the good-hearing group (SD = 2.2) and 17.2 years for the hearing-impaired group (SD = 2.5); t(34) = 0.84, n.s.. The two groups were also similar in vocabulary knowledge as measured by a 20-item version of the Shipley vocabulary test (SD = 2.4) and the hearing-impaired older adults had a mean score of 17.4 (SD = 2.4); t(34) = 0.14, n.s..The good-hearing and hearing-impaired older adult groups were similar in age, with the good-hearing group ranging from 68 to 84 years , all of whom had age-normal hearing acuity, with a mean PTA of 6.7 dB HL (SD = 3.1). At time of testing the young adults had completed fewer years of formal education  than either the good-hearing, t(34) = 3.29, p < 0.01, or the hearing-impaired, t(34) = 4.02, p < 0.001, older adults. As is common in adult aging  than either the good-hearing, t(34) = 4.41, p < 0.001, or the hearing-impaired, t(34) = 4.47; p < 0.001, older adults.For purposes of comparison we also included a group of 18 younger adults , ranging in age from 18 to 29 years  scores for each of the young adults and each of the good-hearing and hearing-impaired older adults taking part in the experiment. The variability within groups and the overlap between groups stands out clearly. Given this variability there was no significant difference between the scores for two older adult groups, t(34) = 0.46, n.s.. Although Figure 2 shows a tendency for the young adults’ distribution to be shifted higher relative to the two older adult groups, the overall difference showed only a non-significant trend as compared with the good-hearing older adults, t(34) = 2.03, p = 0.051, and no significant difference relative to the hearing-impaired older adults, t(34) = 1.42, p = 0.17.short separation) or placed so as to separate the person performing the action and the action being performed by at least four intervening words (long separation). Examples of these six sentence types are illustrated in the previously described Table Preparation of the stimuli began with construction of 144 six-word English sentences with a subject-relative structure. In each sentence a male agent  or a female agent  was performing an action . In half of the sentences the male was the agent of the action and in half the female was the agent. For each of these sentences a counterpart sentence was then constructed using the same words but with the meaning expressed with an object-relative structure. In addition, for each of these subject-relative and object-relative sentences a plausible four-word prepositional phrase was inserted in a position that kept at most a one word separation between the person performing the action and the action being performed  that digitized (16-bit) at a sampling rate of 44.1 kHz. Recordings were equalized within and across sentence types for root-mean-square (RMS) intensity using MATLAB . There were also 48 filler sentences prepared that consisted of six- to nine-word active-conjoined sentences that were similar in content to the test sentences but that did not contain an embedded clause structure.Each participant heard 144 test sentences, 24 in each of the six sentence types  along with the 48 filler sentences. Participants were instructed to listen to each sentence as it was presented and then to indicate whether it was either the male or the female in the sentence that was performing the action. Responses were made by pressing the correct one of two keys labeled male or female.Half of the sentences of each type  were presented to participants at 65 dB HL, a level that approximates everyday conversational speech. The remaining half of each sentence type was presented at 20 dB above each the participant’s better-ear PTA  heard more than once by any participant. Sentences and sound level presentation conditions were counterbalanced across participants such that, by the end of the experiment, each base sentence had been heard an equal number of times in each of its syntactic and agent-action separation versions and at 65 dB HL and 20 dB SL an equal number of times. Sound levels were blocked in presentation, with the order of sound level blocks counterbalanced across participants. Sentence types were randomized in order of presentation within the sound-level blocks. Written informed consent was obtained from all participants according to a protocol approved by the Brandeis University Institutional Review Board prior to the start of the experiment.To insure audibility of the stimuli participants were presented with two sentences at 65 dB HL, and two sentences at 20 dB SL for that individual, with one sentence at each intensity level having a subject-relative structure and one with an object-relative structure. The participant’s task was simply to repeat each sentence aloud as it was heard. None of these sentences was used in the main experiment. The good-hearing older adults had 100% word report accuracy at both 65 dB HL and 20 dB SL. The older adult hearing-impaired group scored a mean of 99.5% correct at 65 dB HL and 100% correct at 20 dB SL. The young adults scored 100% correct at 65 dB HL and 99.8% correct at 20 dB SL.Figure 3 that shows the percentage of correct comprehension responses for subject-relative and object-relative six-word, 10-word short separation and 10-word long separation sentences when heard at 65 dB HL and at 20 dB SL for the three participant groups. Consistent with expectations it can be seen that comprehension of sentences with the syntactically simpler subject-relative structures was excellent for all three participant groups regardless of sound-level condition, sentence length, or prepositional phrase placement. The ceiling and near-ceiling level performance for the subject relative sentences also confirms the basic audibility of sentences heard with both sound-level presentations. Differences in comprehension accuracy begin to appear, however, when the syntactic complexity of the sentences was increased by expressing the meaning with an object-relative structure.The main results are summarized in Figure 3 that included effects of syntactic structure , length manipulation , participant group  and presentation level . Participant group was a between-participants variable; all others were within-participants variables. Because of ceiling effects constraining variance for the subject-relative sentences, we performed all ANOVAs and paired-comparison t-tests on rationalized arcsine transformed data  was conducted on the comprehension accuracy data shown in med data .F = 106.09, p < 0.001, F = 4.00, p < 0.05, The ANOVA confirmed a significant main effect of syntactic structure, reflecting the previously cited common finding of poorer comprehension accuracy for the more computationally demanding object-relative sentences than for the less demanding subject-relative sentences, F = 39.32, p < 0.001, Figure 3, however, this main effect was moderated by a significant Length × Syntactic structure interaction, F = 24.11, p < 0.001, F = 3.24, p < 0.05, Of greater interest, the ANOVA also confirmed a main effect of the sentence length manipulation, F = 9.03, p < 0.01, F = 1.53, p = 0.23, F = 12.53, p = 0.001, Although both presentation sound levels were suprathreshold, as confirmed by the previously cited audibility check, the uniform presentation level of 65 dB HL was relatively louder than the 20 dB SL presentation level for all three participant groups. This difference resulted in a significant main effect of presentation level on comprehension accuracy, We conducted a series of follow-up ANOVAs and paired-comparisons to explore in more detail the factors underlying this pattern of main effects and interactions. Because comprehension accuracy for subject-relative sentences was at or near ceiling for all participants and all conditions, the analyses that follow were conducted on the data for just the object-relative sentences.p < 0.001) and of presentation level (p < 0.05), but no Length × Presentation level interaction (p = 0.65). Follow-up paired comparison testing failed to show a significant difference between the six-word sentences and the 10-word short separation sentences for either the 65dB HL (p = 0.99) or the 20 dB SL (p = 0.15) presentation levels. That is, the significant effect of sentence length was due to the poorer comprehension for the 10-word long separation sentences relative to the six-word sentences and the 10-word short separation sentences for both presentations levels (p levels < 0.05 to < 0.01). The difference in comprehension accuracy for the two presentation levels failed to reach significance for either the six-word sentences (p = 0.12) or for the 10-word short separation sentences (p = 0.43). There was a non-significant trend toward an effect of presentation level for the 10-word long separation sentences (p = 0.053).For the young adults a two-way ANOVA conducted on comprehension accuracy showed a significant main effect of sentence length (p < 0.001) but neither a significant main effect of presentation level (p = 0.45), nor a Length × Presentation level interaction (p = 0.53). Similar to the data for the young adults, paired-comparison testing failed to show a significant difference between the six-word sentences and the 10-word short separation sentences for either the 65dB HL (p = 0.15) or the 20 dB SL (p = 0.15) presentation levels. As with the young adults there was again poorer comprehension for the 10-word long separation sentences relative to the six-word sentences and 10-word short separation sentences for both presentations levels (p levels < 0.01 to < 0.001). The difference in comprehension accuracy for the two presentation levels failed to reach significance for either the six-word sentences (p = 0.94), the 10-word short separation sentences (p = 0.76), or the 10-word long separation sentences (p = 0.20).For the good-hearing older adults a two-way ANOVA conducted on comprehension accuracy showed a significant main effect of sentence length (p < 0.001) and presentation level (p = 0.001). There was no Length × Presentation level interaction (p = 0.51). Although the ANOVA failed to yield a significant Length × Presentation level interaction, planned comparison tests showed no significant difference between the six-word sentences and 10-word short separation sentences at 65dB HL (p = 0.36) but this difference did reach significance with the more challenging 20 dB SL presentation (p < 0.01). The 10-word long separation sentences showed significantly poorer comprehension accuracy than both the six-word and 10-word short separation sentences at both presentation levels (p levels < 0.01 to < 0.001). The difference in comprehension accuracy for the two presentation levels was significant for the six-word sentences (p < 0.05), the 10-word short separation sentences (p < 0.01), and the 10-word long separation sentences (p < 0.01).For the hearing-impaired older adults several of the trends seen for the better-hearing groups were now more marked. A two-way ANOVA conducted on comprehension accuracy for the hearing-impaired participants showed significant main effects of sentence length (p = 0.60), with a trend toward a difference emerging at the 20 dB SL presentation level (p = 0.054). A developing pattern was seen for the 10-word short separation sentences which failed to show a significant difference between the two groups at 65 dB HL (p = 0.64), but a significant difference between the two participant groups did appear for the 20 dB HL presentation level (p < 0.05). For the 10-word long separation sentences there was again no significant difference between groups at 65 dB HL (p = 0.27) but there was a small but significant difference between groups at 20 dB SL (p < 0.05), potentially constrained by the previously noted functional floor of chance level performance for the 10-word long separation sentences with a 20 dB SL presentation level.A final analysis was conducted to compare the two target groups with each other: the good-hearing older adults versus the hearing-impaired older adults. The two groups’ comprehension accuracy was similar for the six-word sentences at the 65 dB HL , hearing acuity , and participants’ chronological age in years. This order was selected to examine any contribution of hearing acuity beyond effects of working memory span, and to determine whether age contributed unique variance after accounting for working memory span and hearing acuity.Table  For each predictor variable in each of the two presentation level conditions we show R2, which represents the cumulative contribution of each variable along with the previously entered variables, and the change in R2, which shows the contribution of each variable at each step. The next column shows the level of significance of each variable and the final column shows the standardized regression coefficients (β). It can be seen that working memory as measured by reading span is a significant predictor of comprehension accuracy for all conditions in the experiment; for both the six- and 10-word sentences and in the latter case for the short and long agent-action separations for both the 65 dB HL and the 20 dB SL presentation levels.The results of the regression analyses are shown in Table 2 also appeared when the data for the young adults were included in the regression analyses.)When the presentation level was at the higher 65 dB HL level, hearing acuity contributed to comprehension accuracy only for the 10-word sentences with a long agent-action separation. When the perceptual task was more challenging in the 20 dB SL condition hearing acuity contributed marginally for the six-word sentences, increasing to a significant contribution for the 10-word short and long separation sentences. That is, hearing acuity contributed significant variance only for the more challenging presentation level and even then only for the longer 10-word sentences. With the contributions of working memory span and hearing acuity taken into account, chronological age did not contribute additional variance to comprehension accuracy. (The same pattern as shown in Although hearing loss is a common accompaniment of adult aging, it has primarily been considered as an independent issue in aging research. There is now a growing recognition, however, that successful speech comprehension reflects an adaptive interaction between sensory and cognitive operations. There are two aspects to this interaction. The first is that the poorer the acoustic quality of the stimulus, whether due to reduced hearing acuity, poorly articulated speech, or the presence of background noise, the more support is required from top-down linguistic knowledge . In the It is the case that all participants, to include those in the older adult hearing-impaired group, successfully scored at ceiling or near ceiling when tested for speech audibility at both sound intensity levels we employed. This should not imply, however, that all groups had access to the same quality of stimulus input. This is the other side of the sensory–cognitive interaction; namely, the previously cited position that successful perception in the face of an acoustically degraded stimulus may come at the cost of resources that would otherwise be available for higher-level cognitive or linguistic operations. This position, in its broad outlines, has sometimes been referred to as an “effortfulness hypothesis” (So long as the processing demands required for sentence comprehension did not exceed an upper limit on total processing resources, as in the case of sentences with a subject-relative structure, successful comprehension was possible even under conditions of perceptual effort. According to this resource argument, this point would have been exceeded when the difficulty in determining the thematic role assignments within a sentence imposed additional processing demands beyond those required for resolution of subject-relative sentences and when greater listening effort was required. This effect was revealed in reduced accuracy for object-relative sentences and when the relational elements were separated by insertion of a prepositional phrase in the long agent-action separation condition. This latter placement would be expected to exacerbate the already greater difficulty in determining thematic roles in object-relative constructions as the relational elements would need to be held in memory for a longer period of time (see The effortfulness hypothesis, which is consistent with extant models that postulate an upper limit on working memory or attentional resources cf., , has somAn additional factor that may be considered can be referred to as an expectancy-uncertainty based account. As noted previously, because object-relative and other syntactically complex forms occur less frequently in one’s everyday listening experience than simpler syntactic forms e.g., , one’s eIt should be noted in this discussion that we do not present working memory and experience-based expectation accounts as mutually exclusive alternatives. Indeed, a study examining eye-movements in reading text has implicated contributions to sentence processing from both sources .Although an expectancy-based account might apply to the traditional finding of greater comprehension errors for object-relative sentences, it would not, in itself, explain why reduced hearing acuity would exacerbate this effect. An expectancy-based account, however, must not only include the likelihood of encountering a particular lexical item or structural form. It must also include an element of uncertainty, sometimes referred to as response entropy see . Here thFirst, it is important to note that the participants in this experiment represented high-functioning older adults with good verbal knowledge and working memory capacity. Indeed, as a group, the good-hearing and hearing-impaired older adults had better vocabulary scores than their younger adult counterparts and a distribution of working memory span scores that were relatively close to that of the young adults. It should also be emphasized that stimuli were presented in quiet, thus avoiding the special difficulty older adults have when hearing speech with background noise . With leSecond, although we have made reference to listening effort, it must be acknowledged that both its definition and measurement remain a topic of debate . It shouDeclines in sensory acuity and efficiency of cognitive function often co-occur in adult aging. Both can affect speech comprehension, with the interaction between the two revealed in the dual challenges of hearing impairment and syntactic complexity in determination of semantic relations in sentence comprehension. It should also be noted that although our focus has been on downstream effects of listening effort, deficits in recall and comprehension of written text with degraded vision have also been reported in the literature . This suAll authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Valuable insights into the role of experience in shaping perception can be obtained by studying the effects of blindness or other forms of sensory deprivation on the intact senses. Blind individuals are particularly dependent on their hearing and there is extensive evidence that they can develop superior auditory skills, either as a result of plasticity within the auditory system or through the recruitment of functionally relevant occipital cortical areas that lack their normal visual inputs. Because spatial processing normally relies on close interactions between vision and hearing, much of the research in this area has focused on the effects of blindness on auditory localization. Although enhanced auditory skills have been reported in many studies, some aspects of spatial hearing are impaired in the absence of vision. In this case, the effects of crossmodal plasticity may reflect a balance between adaptive changes that compensate for blindness and the role vision normally plays, particularly during development, in calibrating the brain’s representation of auditory space. Our perceptions of the world are often determined by the way different sensory modalities interact. In the case of hearing, for example, the ability of human listeners to identify sounds such as speech—particularly against a noisy background—or to judge their locations can be profoundly influenced by the availability of concurrent visual cues  processing. Because of the greater reliance that visually-deprived humans and animals have to place on their intact sensory modalities, it is not surprising that their behavioral performance is often superior to that of sighted controls. Nevertheless, even for the same function, such as sound localization, conflicting results have been obtained. Although some of this variation can be attributed to methodological differences between studies and to differences in the age at onset, duration and severity of blindness, it is clear that certain aspects of spatial hearing can be enhanced, whereas others are impaired in visually-deprived individuals. This likely reflects a trade-off between the lifelong influence of vision in calibrating neural representations of auditory space and the compensatory crossmodal plasticity that results from a combination of reduced inputs in one sensory modality and increased use of the others. In addition, the opposing effects of blindness on localization based on auditory spectral cues in the horizontal and vertical planes suggest that there may be a limited capacity for experience-dependent improvements in neural processing. Further investigation, particularly at a neuronal level, of the location and nature of the changes that take place following blindness will be key to revealing both the mechanisms underlying crossmodal plasticity and the functional and modality specificity of different brain regions.


Current knowledge indicates that the mammalian target of rapamycin (mTOR) functions as two complexes, mTORC1 and mTORC2, regulating cell growth, proliferation, survival, differentiation, and motility. Recently mSin1 has been identified as a critical component of mTORC2, which is essential for phosphorylation of Akt and other signaling molecules. Studies have shown that rapamycin inhibits phosphorylation of mSin1. However, the underlying mechanism is unknown. Here we found that rapamycin inhibited phosphorylation of mSin1 potently and rapidly. Expression of rapamycin-resistant mutant of mTOR (mTOR-T), but not rapamycin-resistant and kinase dead mutant of mTOR (mTOR-TE), prevented rapamycin from inhibiting mSin1 phosphorylation, suggesting that rapamycin-induced dephosphorylation of mSin1 is mTOR-dependent. Surprisingly, ectopic expression of rapamycin-resistant and constitutively active p70 S6 kinase 1 (S6K1) did not confer resistance to rapamycin-induced dephosphorylation of mSin1. Furthermore, disruption of mTORC1 and mTORC2 by silencing raptor and rictor, respectively, or downregulation of S6K1 or Akt did not induce the dephosphorylation of mSin1 as rapamycin did. However, silencing mTOR or mLST8 mimicked the effect of rapamycin, inhibiting mSin1 phosphorylation. Our findings suggest that rapamycin inhibits mSin1 phosphorylation, which is independent of mTORC1 and mTORC2, but is possibly dependent on a new mTOR complex, which at least contains mTOR and mLST8. The mammalian target of rapamycin or the mechanistic target of rapamycin (mTOR), a member of the phosphoinositide-3′ kinase (PI3K)-related kinase family, lies downstream of the type I insulin-like growth factor IGF-1) receptor-PI3K  receptor. StudiesMTOR gene have recently been found to be associated with the hyperactivation of mTOR in tumors  orthophosphate (0.4 mCi/ml)  for 4 h at 37°C. The cells were finally lysed in RIPA buffer , followed by immunoprecipitation with antibodies to mSin1 . The immunoprecipitates were separated by SDS-PAGE and the gel was dried. Phosphorylation of mSin1 (32P-labeled mSin1) was detected by autoradiography. The bands were semi-quantified using NIH image J.Cells, grown in 100-mm dishes, were pretreated with or without rapamycin (100 ng/ml) for 24 h. Subsequently, the used medium was replaced with 3 ml phosphate-free DMEM (Invitrogen), and the cells were then stimulated with or without IGF-1 (10 ng/ml) for 1 h, followed by labeling with [t-test or one-way analysis of variance (ANOVA) followed by post hoc Dunnett's test for multiple comparisons. A level of P < 0.05 was considered to be significant.Results were expressed as mean values ± standard error (mean ± S.E.). Statistical analysis was performed using student


The self-report questionnaire is a popular tool for measuring outcomes in trials of interventions for hearing impairment. Many have been designed over the last fifty years, and there is no single standard questionnaire that is widely accepted and used. We felt it would be a valuable resource to have a comprehensive collection of all adult hearing-loss questionnaires  and to survey their degree of validation. We collated copies of every published hearing difficulty questionnaire that we could find. The search was primarily done by iterative reference searching. Questionnaire topics were obtained by mapping the text of each questionnaire onto a set of categories; reports of validation methods were taken from the primary paper(s) on each questionnaire. In total we found 139 hearing-specific questionnaires (though many others were found that were primarily about something else). Though not formally systematic, we believe that we have included every questionnaire that is important, most of those of some notice, and a fair fraction of those obscure. We classified 111 as “primary” and the remaining 28 as “contractions”, being shortened versions of a primary without any new questions. In total, there were 3618 items across all the primary questionnaires. The median number of items per questionnaire was 20; the maximum was 158. Across all items, about one third were concerned with the person’s own hearing, another third with the repercussions of it, and about a quarter with hearing aids. There was a wide range in validation methods, from only using items chosen statistically from wider pools and with formal validation against independent measures of clinical outcomes, to just reporting a correlation with an audiogram measure of hearing loss. The “state of play” of the field of hearing questionnaires will be discussed.


Etomidate is a rapid hypnotic intravenous anesthetic agent. The major side effect of etomidate is the reduced plasma concentration of corticosteroids, leading to the abnormal reaction of adrenals. Cortisol and testosterone biosynthesis has similar biosynthetic pathway, and shares several common steroidogenic enzymes, such as P450 side chain cleavage enzyme (CYP11A1) and 3β-hydroxysteroid dehydrogenase 1 (HSD3B1). The effect of etomidate on Leydig cell steroidogenesis during the cell maturation process is not well established.Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Srd5a1, and Akr1c14. The testis mitochondria and microsomes from 35-day-old rat testes were prepared and used to detect the direct action of etomidate on CYP11A1 and HSD3B1 activity.Immature Leydig cells isolated from 35 day-old rats were cultured with 30 μM etomidate for 3 hours in combination with LH, 8Br-cAMP, 25R-OH-cholesterol, pregnenolone, progesterone, androstenedione, testosterone and dihydrotestosterone, respectively. The concentrations of 5α-androstanediol and testosterone in the media were measured by radioimmunoassay. Leydig cells were cultured with various concentrations of etomidate (0.3–30 μM) for 3 hours, and total RNAs were extracted. Q-PCR was used to measure the mRNA levels of following genes: 50) values of 12.62 and 2.75 μM, respectively. In addition, etomidate inhibited steroidogenesis-related gene expression. At about 0.3 μM, etomidate significantly inhibited the expression of Akr1C14. At the higher concentration (30 μM), it also reduced the expression levels of Cyp11a1, Hsd17b3 and Srd5a1. In conclusion, etomidate directly inhibits the activities of CYP11A1 and HSD3B1, and the expression levels of Cyp11a1 and Hsd17b3, leading to the lower production of androgen by Leydig cells.In intact Leydig cells, 30 μM etomidate significantly inhibited androgen synthesis. Further studies showed that etomidate also inhibited the LH- stimulated androgen production. On purified testicular mitochondria and ER fractions, etomidate competitively inhibited both CYP11A1 and HSD3B1 activities, with the half maximal inhibitory concentration (IC Etomidate is a carboxylated imidazole first synthesized in 1964 and reported in 1965 by Janssen Pharmaceuticals , 3. Etom50) for cortisol biosynthesis is 1 nM, which is equivalent to the apparent dissociation constant for etomidate binding to membranes of these cells [It has several common side effects, especially for the suppression of endogenous glucocorticoid biosynthesis in adrenal glands due to the inhibition of the important mitochondrial steroidogenic enzyme cytochrome P450 11β-hydroxylase (CYP11B1), which catalyzes the production of cortisol from deoxycortisol. The enzyme is 95% homologous to the aldolase enzyme for mineralocorticoid aldosterone synthesis , 8. Expose cells .There are several common steps for steroid biosynthesis between adrenals and gonads, including initial steps catalyzed by mitochondrial P450 cholesterol side chain cleavage enzyme (CYP11A1) and smooth endoplasmic reticulum enzyme 3β-hydroxysteroid dehydrogenase (HSD3B). Due to these common enzymes and the common substrate, cholesterol, to begin with in both systems, it is highly likely that etomidate may also affect reproductive system. Indeed, after administration with etomidate at 0.25 mg/kg to women, it caused a significant decrease of estradiol, progesterone, 17-hydroxyprogesterone, and testosterone levels within 10 min . In the Cyp11a1), HSD3B1 (encoded by Hsd3b1), 17α-hydroxylase/17,20-lyase , and 17β-hydroxysteroid dehydrogenase 3  as well as testosterone metabolizing enzymes 5α-reductase 1  and 3α-hydroxysteroid dehydrogenase . Cholesterol is the initial substrate used by CYP11A1 and is converted into pregnenolone in the mitochondria [Scarb1). Rat immature Leydig cells are responsive to the luteinizing hormone (LH) secreted by pituitary. When LH binds to luteinizing hormone receptor  to induce LHCGR-complex interaction leading to cAMP cascade, the latter activates protein kinase A, which induces the expression of steroidogenic acute regulatory protein . STAR is the rate-limiting step to transport intracellular cholesterol into mitochondrial inner membrane.The present study will address the effects of etomidate on steroidogenesis in purified male rat immature Leydig cells. The testosterone homeostasis relies on the balance of testosterone biosynthesis and metabolism. Rat immature Leydig cells were good model for the study of testosterone biosynthesis and metabolism, because these cells contain testosterone biosynthetic enzymes: CYP11A1 . Unlabeled pregnenolone, progesterone, 17α-hydroxyprogesterone, androstenedione and testosterone were obtained from Steraloids . Etomidate was purchased from Sigma . Male Sprague-Dawley rats (30-day-old) were purchased from Shanghai Animal Center . All animal procedures were approved by the Institutional Animal Care and Use Committee of Wenzhou Medical University and were performed in accordance with the Guide for the Care and Use of Laboratory Animals.[2. Testes were removed and Leydig cells were purified as described previously [2 tank, testes were removed, perfused with collagenase (0.1 mg/ml) via testicular artery, digested with collagenase (0.25 mg/ml) and DNase (0.25 mg/ml) for 15 min, filtered with nylon mesh, and the cells were separated under Percoll gradient. The cells with density of 1.070–1.088 g/ml were collected and washed. Purities of Leydig cell fractions were evaluated by histochemical staining for HSD3B activity, with 0.4 mM etiocholanolone as the steroid substrate [Eighteen 35-day-old male Sprague Dawley rats were sacrificed by asphyxiation with COeviously . In brieubstrate . The pur6 cells/well. Leydig cells were cultured in 0.5 ml DMEM:F12 medium (phenol-free) without  or with hormone and signalling substances, 10 ng/ml LH and 10 mM 8-bromo-cAMP (8Br-cAMP), 20 μM of various steroid substrates including 25R-OH-cholesterol (25R-OHC), pregnenolone, progesterone, androstenedione, testosterone, dihydrotestosterone for 3 hours in the presence of 30 μM etomidate (etomidate was dissolved in ethanol and ethanol was the control). Because 8Br-cAMP can penetrate the cell membrane, therefore it is used to replace the intracellular cAMP, which is impermeable. 25R-OHC, pregnenolone, progesterone, androstenedione, testosterone, and dihydrotestosterone were used as the respective substrate of the enzyme: CYP11A1, HSD3B1, CYP17A1, HSD17B3, SRD5A1, and HSD3A. Because 25R-OHC can readily penetrate cell and mitochondrial membrane, it is used to replace cholesterol as substrate for CYP11A1. Media were collected for DIOL and testosterone assay after incubation.After isolation, the purified immature Leydig cells were seeded into 24-well culture plated with cell density of 0.05 × 10in vivo testicular (Leydig cells) temperature is 34°C.Mitochondrial, cytosol and microsomal preparations of rat testes were done as described previously . Testes 50), 60 min reactions were initiated by addition of rat (10 μg) testis mitochondrial protein in the presence of varying concentrations of etomidate (0.01–100 μM). In separated assays to determine the inhibitory mechanism of etomidate, concentrations of 25R-OHC ranging from 0.002 to 10 μM were added to reaction mixture containing 10 μg rat testis mitochondria and 10–50 μM etomidate, and the mixtures were incubated at 34°C for 60 min. By end of incubation, the product, pregnenolone, was assayed by RIA kit. The percentage conversion of 25R-OHC to pregnenolone was calculated by pregnenolone from the substrate.CYP11A1 activity in testicular mitochondria was assayed using 25R-OHC as a substrate and pregnenolone as a product. Briefly, 25R-OHC (2 μM) was dissolved in ethanol, with final ethanol concentration in the reaction solution no more than 0.2%. The substrate concentration was selected based on the Km value in a pilot study. In order to determine half maximal inhibitory concentration  were incubated with certain amounts of enzymes  for 60–90 min at 34 C . For some reactions, etomidate was added as inhibitor (up to 100 μM). By the end of incubations, the reactions were stopped by adding 2 ml ice cold ether. The steroids were extracted, and the organic layer was dried under nitrogen. Steroids were separated chromatographically on thin layer plates  in chloroform and methanol , and radioactivity was measured using a scanning radiometer . The perPregnenolone, DIOL and testosterone concentrations in the medium was measured with a tritium-based radioimmunoassay (RIA) as described , using tLhcgr, cholesterol transporting genes including Scarb1 and Star, and steroidogenic enzyme genes including CYP11A1 (Cyp11a1), HSD3B1 (Hsd3b1), CYP17A1 (Cyp17a1), HSD17B3 (Hsd17b3), SRD5A1 (Srd5a1), and HSD3A (Akr1c14). HSD3A, or more commonly, 3α-HSD, is coded by AKR1C4 gene in human. The analogue of AKR1C4 in the rat is Akr1c14. The relative mRNA levels of targeted genes were normalized to Rps16 . The RNA was reversely transcribed into cDNA using random hexamers and MMLV reverse transcriptase by the kit  according to the manufacturer’s instruction. Q-PCR was carried out in a 25-μl reaction volume with SYBR Green detection system . Reactions were run on a Bio-Rad qPCR system  for up to 40 cycles and the melting curves were always checked afterward.Total RNAs were extracted from immature Leydig cells using Trizol reagent  according to the manufacturer's instruction. The ten Leydig cell genes and their primers were used as described previously , 22. The50 was calculated using GraphPad  using nonlinear regression of curve fit with one-site competition. Lineweaver-Burk analysis was used to determine the mode of inhibition. All experiments were repeated 3–5 times, depending on the experiments. All data are expressed as means ± SEM. Differences were regarded as significant at P < 0.05.Data were subjected to analysis by student t-test to identify significant differences whenever two groups (a single concentration of etomidate vs. control) were compared. Data were subjected to analysis by Kruskal-Wallis test followed by ad hoc Dunnett's multiple comparisons to identify significant differences between the tested group and the controls whenever there were three or more groups (multiple concentrations of etomidate vs. control) were compared. The ICThe rat immature Leydig cell primarily produces DIOL, because it contains androgen metabolizing enzymes (SRD5A1 and HSD3A)  Fig 1)..Fig 1). When T and DIOL were considered separately, the results were also intriguing. With P5 and D4 as substrates, T productions were not affected  while thSince etomidate (30 μM) is capable of reducing androgen productions under unstimulated condition , we then tested how the dosages of etomidate may affect androgen productions. With the three concentrations we tested, etomidate inhibited both T and DIOL productions in a dose-dependent manner . The effSrd5a1 level. At the highest concentration (30 μM) tested, etomidate also significantly downregulated Cyp11a1, Hsd17b3 and Akr1c14 levels. However, at this highest concentration, it did not affect the expression levels of Lhcgr, Scarb1, Star, Hsd3b1, and Cyp17a1. These results suggest that etomidate is also capable of interfering specific steroidogenic protein productions.We examined the effects of etomidate on the expression levels of genes that are related with androgen biosynthesis and metabolism . Statist50 values for CYP11A1 and HSD3B1 were 12.62 ± 0.117 μM and 2.75 ± 0.085 μM, respectively  and metabolizing enzyme (SRD5A1 and HSD3A) activities. As shown in ectively , Fig 7.+ was used as substrate, it uncompetitively inhibited the enzyme activity, suggesting that etomidate may only bind to the complex of enzyme-NAD+ complex, instead of enzyme itself . Inhibitions in these key synthetic enzymes surely could contribute to the inhibition of testosterone production by etomidate. However, etomidate was also capable of inhibiting the expression levels of testosterone-metabolizing enzymes (Srd5a1 and Akr1c14). Apparently, the inhibitions in synthetic pathway may play more dominantly role than in the metabolic process because even in the face of reductions in metabolic process, testosterone was still reduced. Overall, these results suggest that etomidate may impact multiple cascades in the steroidogenic pathway, which may contribute to the reductions in both T and the final product DIOL.The testosterone homeostasis in rat immature Leydig cells primary depends on the four testosterone biosynthetic enzymes  and two testosterone metabolizing enzymes (SRD5A1 and HSD3A). In the present study, we found that etomidate can, at one hand, dose-dependently inhibit the activities of two key testosterone synthetic enzymes CYP11A1 and HSD3B1. At another hand, etomidate is also capable of dose-dependently down-regulating the expression levels of the two enzymes  at 3 μM > Cyp11a1 (encoding CYP11A1), Hsd17b3 (HSD17B3) and Alk1c14 (encoding HSD3A) at 30 μM. However, even at the highest concentration tested (30 μM), etomidate had no effects on Hsd3b1 and Cyp17a1 and other important Leydig cell specific genes  that are related to the androgen production. At present, though our study shows that 3 hour exposure is capable of inhibiting the mRNA levels of some of the key steroidogenic enzymes, the evidence that etomidate can affect steroidogenic protein levels is missing. However, it is possible that longer term exposure could result in reductions in the protein levels. For example, in cultured adrenocortical cells, long term  exposure to etomidate was able to reduce the steroidogenic output to less than 10%, well over the simple competitively inhibition of enzyme themselves, suggesting a combination effects of reduction in protein levels and of direct inhibition in enzyme activity of CYP11A1 [The treatment of etomidate also significantly inhibited the expression levels of some testosterone biosynthetic enzymes and metabolizing enzymes. The inhibitory potencies were:  CYP11A1 Etomidate is widely used in clinics. To maintain the ideal anesthesia, a blood level of 300~500 ng/ml (1.22–2.04 μM) is required. When its blood level reaches 150~300 ng/ml (0.61–1.22 μM), etomidate causes sedentary effects. In this concentration range, etomidate apparently is also capable of interfering androgen formations by inhibiting the enzyme activities. Whether such inhibitions might also happen to human boys who contain comparable developmental stages of immature Leydig cells as we used in this study deserves further study.S1 FigRat testis HSD17B3 activity was measured by adding androstenedione (D4) and the formation of product of testosterone (T) by rat testis microsome.(EPS)Click here for additional data file.S2 FigRat testis HSD3B1 activity was measured with various concentration of etomidate (ETO). Lineweaver–Burk plot for HSD3B1 in the presence of NAD+. Mean ± SEM, n = 4.(EPS)Click here for additional data file.


To study the changes in the outer retinal microstructures during a six month period after the onset of acute zonal occult outer retinopathy (AZOOR)-complex by spectral-domain optical coherence tomography (SD-OCT).Seventeen eyes of 17 patients with the AZOOR-complex were studied. The integrity of the external limiting membrane (ELM), ellipsoid zone , and interdigitation zone  were evaluated in the SD-OCT images obtained at the initial visit and at six months. The three highly reflective bands were divided into three types; continuous, discontinuous, and absent. The integrity of the outer nuclear layer (ONL) was also assessed.Among the three highly reflective bands, the IDZ was most altered at the initial visit and least recovered at six months. Fifteen of 17 eyes (88%) had a recovery of at least one of the three bands at six months in the retinal area where the ONL was intact, and these areas showed an improvement of visual field. Three eyes (18%) had retinal areas where the ONL was absent at the initial visit, and there was no recovery in both the retinal structures and visual fields in these areas.Our results indicate that more than 85% eyes with AZOOR-complex show some recovery in the microstructures of the outer retina during a six month period if the ONL is intact. We conclude that SD-OCT is a useful method to monitor the changes of the outer retinal microstructure in eyes with the AZOOR-complex. Acute zonal occult outer retinopathy (AZOOR) is a retinal disease that was first reported by Gass In 2003, Gass suggested that retinal diseases similar to AZOOR, e.g., multiple evanescent white dot syndrome (MEWDS), multifocal choroiditis and panuveitis (MFP), punctate inner choroidopathy (PIC), acute idiopathic blind spot enlargement (AIBSE), acute macular neuroretinopathy (AMN), and AZOOR, were part of a spectrum of a single disease with similar clinical signs, symptoms, and ophthalmological findings Optical coherence tomography (OCT) is a useful method to detect subtle morphological changes in retinas affected by various pathological conditions. Past studies have demonstrated the diagnostic value of time-domain (TD) and spectral-domain (SD) OCT in eyes with AZOOR-complex. The findings showed that the integrity of the external limiting membrane (ELM), ellipsoid zone  measured by a standard Japanese decimal visual acuity chart at 5 m, slit-lamp biomicroscopy, color fundus photography, Humphrey static perimetry (30-2 program), and SD-OCT. Fluorescein angiography was performed only at the initial visit. Full-field electroretinograms (ERGs) or multifocal ERGs were recorded at the initial visit for a correct diagnosis of the AZOOR-complex The procedures used conformed to the tenets of the World Medical Association's Declaration of Helsinki. Mie University Institutional Ethics Review Board approved this retrospective study of the patients' medical records. Written informed consent was not given by participants for their clinical records to be used in this study, but patient information was anonymized and de-identified prior to analysis.All of the patients had undergone SD-OCT examinations with the Spectralis OCT  or the Cirrus HD-OCT . Following the dilation of the pupils, the retinal tomographic images of 9 mm (approximately 30°) horizontal scans for Spectralis or horizontal 6 mm scans for Cirrus were obtained across the fovea. Depending on the image quality, B-scans were averaged.We evaluated the SD-OCT findings at the initial visit and at six months. The reason why we selected the SD-OCT findings at 6 months was because Gass et al. To evaluate the changes in the outer retinal highly reflective bands more quantitatively, a longitudinal reflectivity profile (LRP) was created by previously described methods The clinical characteristics and SD-OCT findings of the 17 Japanese patients with AZOOR-complex  are summarized in A 35-year-old myopic woman presented with complaints of acute vision reduction and photopsia in her right eye. Her decimal best-corrected visual acuity (BCVA) was 0.5 OD, and perimetry showed a visual field defect within 30 degrees of the fovea . Fundus Her SD-OCT findings at the initial visit are shown in At six months after the initial visit, the decimal BCVA had improved to 1.2, and the visual field had recovered at many points . SD-OCT We also noted that this patient had an area of the retina where the ONL was completely absent at the initial visit  was created in the retina of Case 2 . One verA 34-year-old myopic woman reported experiencing photophobia and vision reduction of one month duration in her right eye. She had a history of Basedow disease for eight years. Her decimal BCVA was 0.5 OD. Fundus examination and fluorescein angiography were normal, but Humphrey visual field tests revealed a temporal scotoma extending into the fixation point in the right eye . The mulHer SD-OCT findings of the right eye at the initial visit are shown in The changes in the ELM, EZ, and IDZ at the initial visit and six months later are summarized in We found that the IDZ was most vulnerable among the three outer retinal bands at the initial visit. There were no AZOOR-complex patients who had a continuous IDZ at the area of visual field defect at the initial visit. The IDZ was also most slow to recover at six months after the initial visit. Even at six months, the IDZ was continuous only in three eyes (18%), discontinuous in six eyes (35%), and still absent in eight eyes (47%).When compared to the IDZ, the ELM and EZ were relatively well preserved at six months. The ELM was continuous in 14 of 17 eyes (82%) at six months. Similarly, the EZ was continuous in 15 of 17 eyes (88%) at six months .We also found that 15 of 17 eyes (88.3%) had a recovery of at least one of the three bands during six months  if the OWe investigated the changes in the outer retinal microstructures at the initial visit and six months later in 17 eyes with AZOOR-complex using the SD-OCT. There have been many case series which described the change of OCT findings with time in AZOOR-complex patients We found that the IDZ was most vulnerable among the three outer retinal bands in the retina of AZOOR-complex. At the initial visit, the IDZ was abnormal, i.e., discontinuous or absent, in all 17 eyes (100%), while the ELM and EZ were abnormal in 11 (65%) and 16 eyes (94%), respectively . SimilarThe origin of IDZ has not been established, and is currently thought to correspond to the junction There are several reports suggesting that the abnormality of EZ was present in the region of the visual field defects in AZOOR-complex Spaide et al. Many of our patients with AZOOR-complex had myopia, and the average spherical equivalent refractive error in our 17 eyes was -4.4 D. This is consistent with past reports showing a high prevalence of myopia in eyes with AZOOR-complex There are two major limitations in this study. The first limitation is the small number of patients who were studied retrospectively. Because the AZOOR-complex is a very rare disease, we could not collect many patients from only two institutions. In addition, some patients were excluded because of insufficient SD-OCT or clinical data. Longer prospective studies with a larger number of patients may clarify more detailed information on the structural and functional changes with time in AZOOR-complex.The second limitation is that we have combined the three different subtypes of the AZOOR-complex, MEWDS, AZOOR, and AIBSE. Although Gass et al. Despite these limitations, our results demonstrated that 15 of 17 eyes (88%) with AZOOR-complex have some recovery of the retinal microstructures during six months if the ONL is intact. We also showed that the IDZ was the most vulnerable at the initial visit, and difficult to recover in this disorder. The SD-OCT was very useful for monitoring the changes of the outer retinal microstructure in eyes with the AZOOR-complex.