In the mature eye, three distinct tissue fates, retina, ciliary body, and iris, arrange with a strict linear organization along the central (back) to peripheral (front) axis. The establishment of this topographical relationship within the optic vesicle is not well understood. We use a targeted vital labeling strategy to test the derivation of mature eye tissues from the optic vesicle of the chick embryo. Fate mapping uncovers two distinct origins of the neural retina. Contrary to expectations, the central neural retina has a discrete origin within the posterior optic vesicle. The peripheral retina derives from the distal optic vesicle, sharing a common origin with more peripheral tissue fates. This study identifies for the first time two distinct retinal sub-domains, central and peripheral, which arise during embryogenesis. Identification of these discrete retinal compartments provides a framework for understanding functional and disease processes throughout retinal tissue. The morphogenesis of the vertebrate eye begins as a lateral evagination of the anterior neural tube. As development proceeds, continued lateral expansion creates a distinct optic vesicle that is in contact with the surface ectoderm. During the growth process in the chick, the simple vesicle contorts and shifts, as neural crest and paraxial mesoderm invade the area; this can be observed in transverse sections through the region The mature optic cup is patterned along all three of its axes , nasal-temporal (NT), and central-to-peripheral ). Dorsal-Ventral and Nasal-Temporal patterning influence how the retinal neurons connect to the brain. Central to peripheral pattering in the OC results in distinct tissues , occupying progressively central to peripheral positions within the eye. During development, this axis is observed in the central to peripheral gradient of neuronal maturation in the retina and the difference in neuronal derivatives from central versus peripheral progenitors A lineage boundary has been described along the dorsal-ventral axis of the eye, and is most likely established during optic vesicle stages There are several unique anatomical structures that define the central retina in adult eyes and yet very little understanding of how these features develop. Although there are inter- and intra-species differences , all vertebrates have their area of highest visual acuity in the central retina. This region is often cone-rich, a zone where the neuroblasts have never produced rod photoreceptors. There are human syndromes that preferentially affect the development of central retina/macula, while the peripheral retina develops normally The present study shows that domains giving rise to A) central neural retina, B) central RPE, or C) optic cup lip can be identified in the OV. Contrary to expectations, the distal tip of the optic vesicle gives rise to the OCL and not the central neural retina within the optic cup. Our findings lead to a new fate map of the OV, in which the distal tip of the optic vesicle gives rise to the OCL, and then to the majority of the peripheral retina. Finally, we propose that there are distinct developmental histories, in terms of potential microenvironments and gene expressions, such that the future optic cup lip and thus peripheral retina is a different compartment than the central retina.Fertilized chicken eggs  were obtained from a local breeder  and incubated in a humid incubator at 39°C until Hamburger Hamilton stage (HH) 9/10. For in-ovo injections, embryos were visualized by opening the eggshell and injecting contrast ink below the embryo. Following injection, eggshells were resealed with parafilm  and embryos re-incubated until ages required. For ex-vivo manipulations, embryos were cultured, ventral side up, according to the modified New Culture method Proviral plasmids encoding GFP expression driven by a spleen necrosis virus DiI  and diO  were diluted in tetraglycol . Concentrated virus supernatant was mixed with CellTracker™ Green CMFDA (5-Chloromethylfluorescein Diacetate) (Molecular Probes) to permit confirmation of injection site. Dye or virus solution was back-loaded into pulled glass microcapillary pipettes. The vitelline membrane over the head was opened to expose the optic vesicle, capillary positioned, and dye/virus solution injected using a Harvard Instruments pressure injector to deliver the smallest observable amount to the basal surface of the optic vesicle epithelium. Images were captured to record labeling position and embryos re-incubated for between 36 and 72 hours.Dye labeling was assessed in fixed embryos by capturing images with a Leica MZ16F stereomicroscope fitted with a Jenoptik ProgRes MF camera. Virus embryos were similarly imaged prior to fixation. Images were compiled using Adobe Photoshop v11.0.2 (Adobe Systems) and ImageJ v1.44o . Measurements of dye position were carried out using Image J. For distal OV label, the point where the embryonic midline crossed the longest axis of the optic vesicle was marked, a vector between the dye label in the distal OV and this point drawn, and the angle to the midline measured . For optEmbryo heads were sunk in PBS containing 20% sucrose and embedded in a 1∶1 solution 20% sucrose in PBS: OCT . Blocks were frozen on dry ice. Serial cryosections (10 µm) were collected and processed to visualize dye labeling or for immunohistochemistry. Sections for immunohistochemistry were blocked in PBS: 1% bovine serum albumen: 10% normal goat serum for 30 minutes before incubation in the same solution containing primary antibodies overnight at 4°C, followed by secondary antibody for 1 hour at room temperature. All sections were mounted in DAPI  containing mounting media . Primary antibodies used were mouse anti-Collagen IX clone 2C2 , mouse anti-HuC , rabbit anti-GFP , mouse anti-Lhx1 clone 4F2 , rabbit anti-Lhx2/9 , diluted to 1∶250, 1∶100, 1∶2000, 1∶250, 1∶1000 respectively. Antibody labeling was visualized with complimentary secondary antibodies conjugated with Alexa488 or 594 . Comparable neighboring sections were similarly processed omitting primary antibody to control for non-specific secondary antibody labeling. Images were collected using a Nikon E800 microscope fitted with a Spot RT3 camera.Prevalent models of eye development in higher vertebrates are based on the assumption that terminal eye compartments, that is the neural retina, RPE and the future ciliary body/iris, are largely fixed once the optic vesicle has morphed into a cup OCL label, dye in the optic cup lip plus equal dye in anterior inner and outer layers; Neural Retina label, dye confined to the inner layer; RPE label, dye confined to the outer layer , labeling applied using the same targeting criteria (the junction of the posterior optic vesicle and the contacting mesoderm) labels the temporal OCL; the central neural retina domain has shifted, as expected, towards the surface ectoderm (data not shown). A role for sheet and/or cell movement during the transition from vesicle to cup is possible. Recently, experiments comparing fish and chick eye development highlighted rotational cell movements during OV growth in the fish, and it was proposed that similar movements occur during chick OV development Lineage analysis by vital dye labeling was used by Dutting and Thanos We have concentrated in this study on the dorsal optic cup. Ventral OV labeling was carried out to examine any contribution of the ventral OV to the dorsal OC. Labeling the ventral OV, at the stages used here, distributed to the optic stalk/presumptive choroid fissure and brain but never to the dorsal OC (data not shown). Our preliminary observations indicate that the ventral OC is formed later than the dorsal, from cells that occupy the choroid fissure, and which originated in the ventral OV.Whether our fate map of the avian eye is directly transferable to understanding eye growth in the mammalian eye remains open to testing. The direct lineage analysis presented here is not easily done in the mammalian system. However, in support of conserved mechanisms, cre-driven lineage analysis in the eye has been interpreted as presenting distinct origins for peripheral and central retina We have uncovered distinct OV origins of central and peripheral eye tissues, revising earlier models of eye morphogenesis . We propThe peripheral eye originates at the distal OV. Dye labeling shows that the total of the labeled spots comprise a field of cells that form into the OCL around the dorsal eye. As with direct OCL targeting While timing of labeling almost certainly allows distinct central OC precursor to be identified, more importantly, it highlights differences in embryonic history that may contribute to overt functional differences between central and peripheral retina: central retina emerges early from a common eye niche and is never incorporated into the OCL niche that distributes progeny throughout the remainder of the eye. Further study is required to understand both external patterning that acts upon central and peripheral eye domains leading to fate determination, and the intrinsic cell changes that may reflect timing of emergence from a common progenitor niche. The distinct micro-environments that central RPE and neural retina precursors occupy in the optic vesicle allow speculation of adjacent tissues that may be involved in fate assignment and differences in protein expression will likely allow perturbation of these compartments. Similarly, examination of differences in cell potential along the proximo-distal axis of the optic vesicle may unveil stepwise commitment of cells towards a particular eye fate. With the resolution afforded by the finite unbiased fate tracing herein, stringent interpretation of optic vesicle perturbation is possible. The demarcation of two distinct retinal domains, that are established during embryogenesis, will allow renewed evaluation of human diseases that are specific to either retinal sub-domain.


Adult-onset hearing loss is insidious and typically diagnosed and managed several years after onset. Often, this is after the loss having led to multiple negative consequences including effects on employment, depressive symptoms, and increased risk of mortality. In contrast, the use of hearing aids is associated with reduced depression, longer life expectancy, and retention in the workplace. Despite this, several studies indicate high levels of unmet need for hearing health services in older adults and poor use of prescribed hearing aids, often leading to their abandonment. In Australia, the largest component of financial cost of hearing loss (excluding the loss of well-being) is due to lost workplace productivity. Nonetheless, the Australian public health system does not have an effective and sustainable hearing screening strategy to tackle the problem of poor detection of adult-onset hearing loss. Given the increasing prevalence and disease burden of hearing impairment in adults, two key areas are not adequately met in the Australian healthcare system: (1) early identification of persons with chronic hearing impairment; (2) appropriate and targeted referral of these patients to hearing health service providers. This paper reviews the current literature, including population-based data from the Blue Mountains Hearing Study, and suggests different models for early detection of adult-onset hearing loss. Adult-onset hearing loss is a highly prevalent yet relatively underrecognised health problem in the older adult Australian population , 2. Becaper se, but also the impact that it has on the individual in terms of activity limitations (such as inability to perceive speech in noisy environments) and participation restrictions  [Hearing loss is a chronic problem and, contrary to current community perception and funding models of hearing services, hearing aids are typically a part of a rehabilitation program rather than provide a single and simple restorative solution to hearing loss . As suchivities) . Nonetheivities) –20. Impoivities) . Despiteivities) , 23 indiivities) . To date, the Australian public health system does not have an effective and sustainable hearing loss screening strategy for late-onset hearing loss in adults to manage this problem. This paper aims to review the current pathway of detection, referral, and management of late-onset adult hearing loss in Australia and to identify an alternative, more effective pathway for the future. 0.5–4 kHz) in the better ear. Any hearing loss was defined as PTA0.5–4 kHz > 25 dBHL. Risk factors measured (either via self-report or practitioner measurement) included self-reported health, noise exposure, and family history of hearing loss. In this study, we identified that a 33.0% prevalence of bilateral hearing loss existed in persons aged 50+ years (51% showed hearing loss in the worse ear) consistent with that measured in the US-based Epidemiology of Hearing Loss Study (EHLS) [Australian population-based data describing prevalence, incidence, and risk factors for hearing loss have been identified in the Blue Mountains Hearing Study (BMHS) in 1997–2000 among 2956 participants of the Blue Mountains Eye Study (BMES) cohort  , 26. Of y (EHLS) . More spy (EHLS) . The oveP = 0.025; decrease in mental component score (MCS) of 1.0 point, P = 0.13); with poorer scores associated with more severe levels of impairment  [P < 0.001) [As well as health-related influences, our epidemiological study also assessed quality-of-life and mental health factors, such as cognitive function and depression. BMHS-I data showed that bilateral hearing loss was associated with poorer SF-36 scores in both physical and mental domains  of 1.4 points, < 0.001) . TherefoStephens et al.  suggest Low rates for use of hearing services and hearing aids highlight barriers including cost  and/or rThere remains a large proportion of hearing-impaired adults who would benefit from hearing aids but who decide not to seek help . FurtherThere exists a need for a readily accessible screening test assessing hearing disability which could more accurately identify rehabilitation need, rather than just measurement of hearing loss. Validated, self-administered questionnaires about hearing disability have been shown to detect functional hearing impairment accurately and, so, have been recommended as potential screening tools –52. ThesGiven the pivotal role of the GP in the early identification and management of chronic health problems, at least in Australia, the implementation of a GP-based hearing screening program for adults >50 years of age would be beneficial in addressing this problem. Further, with the inadequacies of the medical model in the treatment of chronic health conditions and the move towards a model of patient-centred care, GPs are effectively placed to assist with the minimisation of the stigma associated with hearing loss and enhancing patient self-motivation to manage this . CurrentGiven the increasing prevalence and disease burden of undetected hearing loss in older adults and the availability of effective interventions , there are 3 potential critical roles for the GP in hearing health: (1) early identification of patients with age-related hearing loss, as well as recognition of whether any negative consequences/disability has resulted; (2) assistance in reducing the stigma of hearing loss and motivating patients to seek further help; and (3) appropriate referral of these patients to hearing health providers. This could be achieved by sensitising GPs to recognise at-risk individuals and providing targeted questions to identify hearing loss disability. We have identified an important role of GPs in the process of targeting individuals with late-onset hearing loss and referral; however, the challenge that remains is how to effectively increase GPs knowledge and practice behaviour in this area. Possibly the most obvious method is through development of a continuing medical education (CME) program that targets the impacts of hearing loss and remediation and provides a reliable method of hearing screening in adults. The evidence for good outcomes of CMEs measured by factors including increased knowledge and skills as well as altered attitudes and practice behaviours is varied and possibly depends partly on the learners and learning context . A revier = 0.77, P < 0.001) and Telscreen and the presence of subjectively rated disability . An alternative method of screening of hearing loss and disability which does not require GP involvement is telephone and/or Internet screening using digits in noise –64, whicSmits and colleagues  demonstrAnother hearing screening program is the use of an automated face-to-face monosyllabic speech-in-noise test which aims to evaluate hearing disability in adults. The speech understanding in noise (SUN) test was developed by Paglialonga and colleagues , 67 and Given the ageing demographic and increasing average life span in Western countries, chronic hearing loss is projected to increase. A renewed focus on targeting the provision of hearing rehabilitation to people with self-perceived hearing disability, rather than those with only measured hearing loss, may lead to better long-term retention and use of aids. Therefore, over time the costs saved by provision of an effective and better-targeted health intervention enabling improved daily functioning among older adults will no doubt demonstrate this strategy and will provide “value for money.”


Eimeria tenella in the chicken intestine is responsible for avian coccidiosis, a disease that has major economic impacts on poultry industries worldwide. E. tenella is transmitted to naïve animals via shed unsporulated oocysts that need contact with air and humidity to form the infectious sporulated oocysts, which contain the first invasive form of the parasite, the sporozoite. Cysteine proteases (CPs) are major virulence factors expressed by protozoa. In this study, we show that E. tenella expresses five transcriptionally regulated genes encoding one cathepsin L, one cathepsin B and three cathepsin Cs. Biot-LC-LVG-CHN2, a cystatin derived probe, tagged eight polypeptides in unsporulated oocysts but only one in sporulated oocysts. CP-dependant activities were found against the fluorescent substrates, Z-FR-AMC and Z-LR-AMC, throughout the sporulation process. These activities corresponded to a cathepsin B-like enzyme since they were inhibited by CA-074, a specific cathepsin B inhibitor. A 3D model of the catalytic domain of the cathepsin B-like protease, based on its sequence homology with human cathepsin B, further confirmed its classification as a papain-like protease with similar characteristics to toxopain-1 from the related apicomplexan parasite, Toxoplasma gondii; we have, therefore, named the E. tenella cathepsin B, eimeripain. Following stable transfection of E. tenella sporozoites with a plasmid allowing the expression of eimeripain fused to the fluorescent protein mCherry, we demonstrated that eimeripain is detected throughout sporulation and has a punctate distribution in the bodies of extra- and intracellular parasites. Furthermore, CA-074 Me, the membrane-permeable derivative of CA-074, impairs invasion of epithelial MDBK cells by E. tenella sporozoites. This study represents the first characterization of CPs expressed by a parasite from the Eimeria genus. Moreover, it emphasizes the role of CPs in transmission and dissemination of exogenous stages of apicomplexan parasites.The invasion and replication of  Eimeria spp. are enteropathogens that infect a variety of mammals and birds. Avian coccidiosis is caused by the infection of chicken with extremely well adapted Eimeria spp. These infectious diseases lead to major economic losses worldwide in poultry industries. Anticoccidial drugs are used extensively to control the disease but the increase of resistant parasite populations underlines the need to find alternative targets and drugs.Eimeria, belongs to the phylum Apicomplexa, a group of medically and economically important parasites including Plasmodium spp.  and Toxoplasma gondii (the agent of toxoplasmosis). Among the seven species of Eimeria commonly detected in infected chickens, Eimeria tenella is one of the most virulent and the only one for which the genome has been sequenced (http://www.genedb.org/Homepage/Etenella) E. tenella is divided into an intestinal and an environmental stage. The intestinal stage involves the invasion and replication of parasites within epithelial cells of the chicken intestine, followed by production of male and female gametes, fertilisation and formation of unsporulated oocysts, which are released into the environment. The environmental stage involves maturation, also called sporulation, of unsporulated oocysts into infectious sporulated oocysts. Each sporulated oocyst contains eight haploid sporozoites, the first invasive form of the parasite.The genus, http://merops.sanger.ac.uk/cgi-bin/clan_index?type=P). Thus, several studies have revealed the crucial roles of cysteine cathepsins (including cathepsin B- and L-like proteases) in cell invasion falcipain-1 knock-out strain of Plasmodium falciparum is not affected in its capacity to replicate within the erythrocyte but is impaired in its capacity to produce oocysts Eimeria, an orthologue of a T. gondii cathepsin B, toxopain-1 Cysteine cathepsins related to papain-like enzymes,  are major virulence factors expressed by parasites, including apicomplexan parasites , one cathepsin L , and three cathepsin Cs  were identified using BLASTP to search the E. tenella genome database (http://www.genedb.org/Homepage/Etenella) using, as queries, the putative homologs described in other apicomplexan parasites, namely T. gondii and P. falciparum.Five putative CP genes including one To begin addressing the biological roles of these proteases, we examined their expression patterns by RT-PCR on total RNAs from three different time points of oocyst sporulation: unsporulated (0 h of sporulation); partially sporulated (6 h of sporulation); and fully sporulated (48 h) . ExpressE. tenella , human cathepsin B and chicken cathepsin B was performed  identified by signalP, a predicted prodomain (from Met22 to Pro223) and a catalytic domain (from Leu224 to Leu512). The prodomain is much longer (214 amino acids) than mammalian and bird cathepsin B, as also reported for toxopain-1 E. tenella cathepsin B contains the two archetypal residues (Cys266 and His445) that form the thiolate-imidazolium ion pair and are involved in the nucleophilic attack of the peptidyl substrate as well an asparaginyl residue (Asn465) reported to critically support the catalytic mechanism. C266 is embedded in the highly conserved peptide sequence, CGSC266WAF. Specific cathepsin B motifs are also found in the E. tenella sequence including the G308CXGG motif as well as the residues that form the occluding loop, which is characterized by two adjacent histidine residues  and encompasses amino acid residues Pro344 to Cys371. In addition to its endopeptidase activity, the presence of the occluding loop confers, specifically to cathepsin B, a dipeptidyl carboxypeptase activity We cloned and sequenced the gene encoding the erformed . The E. E. tenella cathepsin B was determined by molecular replacement using the structure of the catalytic domain of human cathepsin B, with which it shares 43.1% sequence identity. Eimeripain exhibits the typical papain-like fold that is representative of the cysteine cathepsin family . It is composed of two domains, referred to as the left (L-) and right (R-), in accordance with the standard view and nomenclature  provided by the right domain , which block the active site cleft on the primed binding site (beyond S'2 subsite) and are responsible for the dipepdityl carboxypeptidase activity of cathepsin B. A comparison of the homology-based model with human cathepsin B shows a high degree of similarity (rmsd 0.49 Å for 245 Cα atoms). The major structural changes in backbone superposition correspond to additional residues and localize at the surface exposed loops including residues Asn334-Ser341, Glu377-Lys383 and Asp389-Thr395. We have, based on its susceptibility to specific inhibitors, its sequence homology and the predicted 3D structure, named the E. tenella cathepsin B, eimeripain.The structure of the t domain . The E. E. tenella allows, for the first time, consideration of knock-in and knock-out strategies to determine the function of genes in the biology of the parasite eimeripain gene. In a first set of experiments, we looked at the localization of the fusion protein within the unsporulated and sporulated oocysts , we found five genes encoding for putative cysteine proteases: one homolog to cathepsin B (eimeripain); one to cathepsin L (EtCPL); and three to cathepsin C . The gene predictions did not allow us to identify the start and stop codons for all genes; however, we were able to determine their expression profiles by using specific primers for conserved regions and were able to show that all these predicted genes are transcribed. Although five cysteine protease genes were identified in the genome of E. tenella, the probe Biot-LC-LVG-CHN2 detected up to eight polypeptides in the unsporulated oocyst. The probe might have detected other cysteine proteases whose genes are specific to the Eimeria genus or multiple forms of the same protease.CPs are major virulence factors for protozoan parasites. They are implicated in key steps of infectious processes making them attractive therapeutic targets  – are expressed in unsporulated oocysts and at the earliest time point of sporulation analyzed in this study (6 h) but not at later time points or in fully sporulated oocysts. This suggests a role(s) in the initiation of sporulation, a component of the parasite's sexual cycle where meiotic division of the diploid zygote, within the unsporulated oocysts, leads to the production of four haploid sporoblasts. However, this idea awaits demonstration since all of the detected protease activity against Z-FR-AMC and Z-LR-AMC in oocyst lysates can be inhibited by the cathepsin B-specific inhibitor, CA-074. Z-FR-AMC and Z-LR-AMC are specific substrates for CPs with endopeptidase activities , so cathepsin Cs, which exhibit dipeptidyl aminopeptidase activities, are not expected to cleave these substrates. However, the same argument cannot account for the absence of cathepsin L-specific activity on Z-FR-AMC or Z-LR-AMC; this may reveal a specific biochemical property of EtCPL or indicate that it is maintained in an inactive pro-form in unsporulated oocysts.2. The E. tenella cathepsin B shares 43.1% sequence identity with the catalytic domain of human cathepsin B and 53% identity with toxopain-1, the cathepsin B from the related apicomplexan parasite, T. gondiiE. tenella cathepsin B has the active triad sequence of cysteine, asparagine and histidine and, like toxopain-1, contains a characteristic occluding loop sequence and has a relatively long pro-domain. Homology modelling reveals that the overall structure of the catalytic domain is very similar to that of human cathepsin B, which in turn is very similar to that predicted for toxopain-1 E. tenella cathepsin B, eimeripain.We identified a single gene coding for a cathepsin – a cathepsin B – that is expressed throughout sporulation. Activity associated with this enzyme corresponds to a 33 kDa protein band detected in all oocyst stages by the probe Biot-LC-LVG-CHNE. tenella. Unsporulated oocysts express the fusion protein, eimeripain_mCHER, uniformly in the zygote. In contrast, eimeripain_mCHER has a punctated distribution in sporozoites. This localization is similar to that described for cathepsin L in T. gondii (TgCPL). TgCPL is associated with endocytic organelles, most particularly a newly reported vacuolar compartment at the apical region of tachyzoites that undergoes dynamic fragmentation during T. gondii replication Eimeria.Studies performed on trafficking of human cathepsin B have shown that a C-terminus GFP fusion of the protease is correctly trafficked and targeted to lysosomal compartments in thyroid epithelial cells E. tenella  cells Experimental protocols were designed in compliance with French law (Décret 2001-464 29/05/01) concerning the use of laboratory animals. Care and euthanasia of animals were practised according to national ethic guidelines and approved by the ethics committee of the Région Centre (CL2007-36). The authors are committed to the principals of the 3Rs: Reduction, refinement and replacement of experimental animals.E. tenella genome sequences and gene models were downloaded from http://www.genedb.org/Homepage/Etenella The genome of E. tenella Houghton was produced by the Parasite Genomics Group at the Wellcome Trust Sanger Institute (http://www.sanger.ac.uk/research/projects/parasitegenomics/) and has been provided prepublication. BLASTP search was performed in GeneDB using as queries CPs described in T. gondii including one cathepsin B, toxopain-1 (AAL60053), one cathepsin L, TgCPL (ABD64744), and three cathepsin Cs, TgCPC1 (AAZ15654), TgCPC2 (AAZ15655) and TgCPC3 (AAZ15656); and P. falciparum including falcipain-1 (AAN37166), falcipain-2 (AAK06665), falcipain-2′ (AAX77225), falcipain-3 (AAF77192), DPAP1 (AAN35758), DPAP2 (AAN36542) and DPAP3 (XP_001351359). CP genes in Eimeria have been named after their closest homologs in T. gondii.7), taken at different time after the commencement of sporulation, were vortexed with 0.5 mm glass beads until the total destruction of oocyst walls. Total RNAs were obtained by treatment of broken oocysts with TRIzol (1 ml) (Invitrogen) for 5 min at room temperature, extraction with 20% chloroform, and precipitation with 50% isopropanol. RNAs were resuspended in nuclease-free water to a final concentration of 1 mg/ml. Aliquots of 1 µg of total RNAs were used to reverse transcribe total mRNAs using a Poly dT primer and reverse transcriptase II (Invitrogen). Segments of cDNAs were amplified using specific primers to eimeripain, 5′-CTGGGGAAGCCGGCCAGCTGTTAGC-3′ and 5′-CGTTCCAGCTGTTCACAGCTAGCCAG-3′, to etcpl, 5′-CAACCAACAAGGTCACTCTTAC-3′ and 5′-CCCTCGAGGGCCCCCGTGCTCG-3, to etcpc1, 5′-CGCCAAAGGCTACGGCTACGTGG-3′ and 5′-CCGCGAGTGAGGTCGGGGTCAATG-3′, to etcpc2, 5′-CCAAATCAGGGCGACTGCGGCTCTTG-3′ and 5′-GCCCCCAACATAATACCATTTGGAAAC-3′, to etcpc3, 5′-CGCTCAGGAGTACAACTACGTGGGTGG-3′ and 5′-GCTGCTGCACAAGCAGCGCTGCTCTGCC-3′, to actin, 5′-ATCTTCATCGTAGACGGAGCCAG-3′ and 5′-GTGTGTCCCACACTGTTCCTATC-3′. The reaction conditions for RT-PCR were 95°C for 1 min, 58°C for 30 s and 72°C for 1 min for 30 cycles using the Go Taq DNA polymerase (Promega). Amplification products were loaded onto a 0.7% agarose gel stained with ethidium bromide.Pellets of oocysts (5×107) were removed at different time after the commencement of sporulation and lysates were obtained by vortexing pellets with 0.5 mm glass beads until the total destruction of oocyst walls 2trans-(propylcarbamyl)oxirane-2-carbonyl)-L-isoleucyl-L-proline (CA-074) for 10 min at 30°C in buffer A and 2 mM DTT before addition of either 10 µM Z-FR-AMC (benzyloxycarbonyl-Phe-Arg-(7-Amido-4-methylcoumarin)), 10 µM Z-LR-AMC ((benzyloxycarbonyl-Phe-Arg-(7-Amido-4-methylcoumarin)) or 10 µM Z-RR-AMC ((benzyloxycarbonyl-Arg-Arg-(7-Amido-4-methylcoumarin)) (Bachem). Activities were monitored on a spectrofluorimeter  with λexcitation = 350 nm, λemission = 450 nm. Independently, lysates were preincubated or not for 1 h at 25°C with 100 µM CA-074 before addition of 20 µM of the cystatin-derived activity-based probe Biot-LC-LVG-CHN2 for 1 h at 25°C in buffer A with 2 mM DTT. Samples were loaded on a 10% SDS-polyacrylamide gel, transferred onto a nitrocellulose membrane  and tagged active CPs were revealed by Western blot using streptavidin coupled to peroxidase (1/3000). Detection was performed using the Supersignal West Pico Chemiluminescent Substrate (Thermo Scientific).Oocysts (5×10E. tenella genome against toxopain-1 (cathepsin B in T. gondii), we obtained a hit with gene ID ETH_00003570 . Primers were designed upstream from the predicted start site (fETCPB3: 5′-GCCAACCACAGCGCAGCTCCGAGGTG-3′) and downstream from the predicted STOP codon (rETCPB3: 5′-GCCGCGGTTAGCCGCAACTTCGACC-3′) to amplify the cathepsin B gene (eimeripain) from E. tenella sporozoite cDNAs. The amplification product was sequenced, which confirmed the sequence prediction of ETH_00003570. To localize eimeripain within the parasite, we constructed a vector allowing the expression of an eimeripain_mCHER fusion under the control of the eimeripain promoter. Since eimeripain does not contain any intron, we used genomic DNA to amplify a 3 kb region with primers fPrEtCPBMlu and rEtCPBSal. The amplification product, MluI-PrEtCPB/EtCPB-SalI, started 1 kb upstream from the start codon of eimeripain  until the last amino acid codon of eimeripain. MluI-PrEtCPB/EtCPB-SalI was cleaved by MluI and SalI and cloned into the mCherry core construct-1 kindly provided by Dr Damer Blake mic-1 promoter, which was replaced by the 3 kb MluI-PrEtCPB/EtCPB-SalI insert, leading to the vector pEimerip/Eimerip_mCherry in which eimeripain is C-terminally fused in frame with mCherry. The fusion protein eimeripain-mCHER is expressed under the control of the endogenous eimeripain promoter.By blasting the http://www.ebi.ac.uk/Tools/msa/clustalw2/). An automated comparative protein modelling program, SWISS-MODEL, was used to predict the 3D structure of the eimeripain catalytic domain The protein sequences for pro-eimeripain (Accession n° JN641867), pro-toxopain-1 (AAL60053), pro-human cathepsin B (AAC37547.1) and pro-chicken cathepsin B (AAA87075.1) were aligned by using CLUSTALW2  as described previously 6 parasites) for REMI transfection 5 transfected sporozoites per animal) via the cloacal route as described previously Freshly excysted sporozoites were resuspended in complete cytomix buffer  and air dried on a glass slide or incubated with MDBK cells for 24 h. Extracellular parasites and infected cells were fixed in 2.7% PFA for subsequent immunofluorescence analysis. Cell and parasite nuclei were fixed and stained with 1.5 µg/ml of DAPI (Vectashield). Fluorescence imaging was done using a Zeiss Axiovert 200 microscope equipped with the Apotome imaging system. Images were generated and analyzed by using the Axiovision Software (Carl Zeiss SA).E. tenella sporozoites pre-incubated with CA-074 methyl ester (CA-074 Me), a specific cell-permeable inhibitor of cathepsin B, was assessed using acridine orange and ethidium bromide, as described previously 5) were incubated with various concentration of CA-074 Me for 1 h at 41°C. Parasites were washed twice in PBS and acridine orange and ethidium bromide were added at 0.1 µg/ml. Extracellular parasites were visualized and counted using an Olympus BX41 microscope. The percentage of non viable parasites was determined as the ratio of red dead parasites versus the entire green population ×100.Viability of extracellular 5) were incubated with non lethal concentrations of CA-074 Me for 1 h at 41°C in 500 µl of Ham's F12. Sporozoites were washed twice in PBS and incubated with MDBK cells in 24 well plates with coverslips at the bottom, (multiplicity of infection of one), for 1 h at 41°C, 5% CO2. Infected cells were washed and fixed in 2.7% PFA. Monolayers were then permeabilized with 0.1% Triton X-100, and intracellular parasites were labelled with a mouse anti-sporozoite antibody (1/2000) followed by incubation with a secondary anti-mouse antibody coupled to Alexa 594 (1/1000) (red fluorescence). Monolayers were mounted in Vectashield containing 1.5 µg/ml of DAPI  to label nuclei. Infected cells were examined using a Zeiss Axiovert 200 microscope. The percentage of intracellular parasites was determined as the number of intracellular parasites (red) for 100 MDBK cells (blue nuclei). More than 200 cells were counted for each condition in three independent experiments with two wells per condition, and the values reported are means ± standard errors.Freshly excysted purified sporozoites (2×10Statistical comparisons, in the viability test or the invasion assay, were performed using first a generalized linear model and then a Newman-Keuls test. Statistics were conducted assuming equal variance, unpaired samples, and using a 2-tailed distribution. For statistical analysis of sporozoite viability prior to the invasion assay, we used a one-way analysis of variance.Differences were considered to be statistically significant at a P value<0.05.


Neural systems must weight and integrate different sensory cues in order to make decisions. However, environmental conditions often change over time, altering the reliability of different cues and therefore the optimal way for combining them. To explore how cue integration develops in dynamic environments, we examined the effects on auditory spatial processing of rearing ferrets with localization cues that were modified via a unilateral earplug, interspersed with brief periods of normal hearing.In contrast with control animals, which rely primarily on timing and intensity differences between their two ears to localize sound sources, the juvenile-plugged ferrets developed the ability to localize sounds accurately by relying more on the unchanged spectral localization cues provided by the single normal ear. This adaptive process was paralleled by changes in neuronal responses in the primary auditory cortex, which became relatively more sensitive to these monaural spatial cues. Our behavioral and physiological data demonstrated, however, that the reweighting of different spatial cues disappeared as soon as normal hearing was experienced, showing for the first time that this type of plasticity can be context specific.These results show that developmental changes can be selectively expressed in response to specific acoustic conditions. In this way, the auditory system can develop and simultaneously maintain two distinct models of auditory space and switch between these models depending on the prevailing sensory context. This ability is likely to be critical for maintaining accurate perception in dynamic environments and may point toward novel therapeutic strategies for individuals who experience sensory deficits during development.  •Ferrets reared with a unilateral hearing loss are able to localize sounds accurately•Adaptation relies on cue reweighting that reverses when normal hearing is available•Auditory cortical neurons show corresponding context-specific plasticity•Contextual cue reweighting maintains perceptual stability in dynamic environments Although experience is critical for shaping the way sensory systems develop, it is unclear how they respond to environmental conditions that change over time. In the case of spatial hearing, developmental plasticity has been studied primarily by inducing a stable hearing loss in one ear, a manipulation that alters the binaural cues used for sound localization . This woIt is unclear, however, what happens when the developing auditory system experiences an intermittent hearing loss in one ear. This is particularly relevant to the development of spatial hearing, since recurring periods of hearing loss are extremely common in infancy . From a One solution to this problem would be to rely more on cues that remain unchanged over time. Under normal hearing conditions, the location of a sound in the horizontal plane is determined primarily by interaural level differences (ILDs) and interaural time differences (ITDs) , both ofTo address this issue, we reared ferrets with a recurring hearing loss in one ear and obtained behavioral and neurophysiological measures of spatial processing from the same animals. Our results demonstrate that the auditory system can indeed switch between different strategies for localizing sound depending on the sensory context.We reared ten animals from the age of hearing onset with an earplug in the left ear but allowed them relatively brief, intermittent periods of normal hearing. Earplugs delay and attenuate sounds in a frequency-dependent manner , alterinWe also measured localization performance in three juvenile-plugged animals using broadband stimuli in which spectral cues were disrupted by introducing varying amounts of spectral randomization, a manipulation that has a negligible effect on the accuracy of azimuth judgments by human listeners with normal binaural hearing but that should make monaural localization much more difficult . In the If juvenile-plugged animals rely solely on spectral cues to localize sound, then their susceptibility to spectral randomization should remain when the earplug is removed. Alternatively, if these animals can switch between different cues, then randomization should have little effect once balanced binaural inputs are provided. Consistent with this latter hypothesis, we found that the effects of spectral randomization were much less pronounced without an earplug  of both juvenile-plugged ferrets and controls A. BecausWe constructed tuning curves by measuring the effect of changing the value of individual cues on the mean firing rate of the neurons. Introducing a virtual earplug dramatically altered the tuning curves for some of these units C but hadWe next constructed a weighting index that compared the MI values obtained using the monaural spatial cues provided to the nonoccluded right ear with the sum of the MI values obtained using the other available spatial cues. The index was designed so that its value would increase if a neuron were to become relatively more sensitive to the right-ear monaural spatial cues. Consequently, our behavioral data predict that this neural weighting index should be higher in juvenile-plugged animals than in controls, but only when a virtual earplug is experienced.Our A1 data conformed to precisely this pattern E. Thus, As predicted by our behavioral  and modeOur goal in this study was to understand how spatial hearing develops when ferrets experience acoustical inputs that change over time, focusing in particular on the effects of an intermittent hearing loss in one ear. In contrast to the poor performance of acutely plugged controls, ferrets reared with one ear occluded acquired the ability to localize sounds accurately while wearing an earplug, and did so by becoming more dependent on the unchanged spectral cues provided by the intact ear. Surprisingly, however, when these animals were subsequently provided with normal inputs to each ear, they became indistinguishable from controls. The mechanism of experience-dependent adaptation therefore appears to involve a context-specific reweighting of different cues, which allows accurate sound localization to be maintained under both normal and abnormal hearing conditions.These findings contrast with the results of previous developmental studies of unilateral hearing loss, which show pronounced changes in spatial processing following the restoration of normal hearing . One impsame cues in different ways depending on the acoustical context and provide the first evidence linking this type of behavioral plasticity with changes in neuronal responses, in particular those of cortical neurons located in the hemisphere ipsilateral to the affected ear. Although it is conceivable that alternative weighting strategies might be implemented by different sets of neurons, recent work suggests that multiple sensory cues can be multiplexed onto the same sets of neurons [If the shape of their external ears is modified by wearing molds, adult humans can learn to unambiguously map auditory space onto two different sets of spectral cues . Our res neurons , suggestPrevious studies have shown that when perceptual judgments are based on cues from different sensory modalities, the greatest weight is given to those cues that provide the most reliable information . To the Although reweighting of spectral cues represents a useful strategy for adapting to changes in auditory inputs, adaptation can also be achieved by remapping altered binaural cues onto appropriate spatial locations. Indeed, this is the way developing barn owls compensate for the effects of monaural deprivation . In contAlthough previous work has emphasized the importance of spectral cues for vertical localization , these cAn involvement of A1 in the flexible processing of spatial cues is consistent with recent work showing that cortical response properties can change depending on the behavioral context in which sounds are presented . Our resGiven that spectral cues are thought to play a minimal role in horizontal localization , an unexIn summary, our results show that the auditory system can adapt to an intermittent hearing loss in one ear, and does so by becoming more dependent on the preserved monaural spatial cues provided to the intact ear. Importantly, the context-specific nature of this adaptive plasticity also allows accurate sound localization to be maintained whenever normal acoustical input is available. This is precisely the sort of mechanism that would be required to protect against the long-term deleterious effects associated with otitis media with effusion, a clinical disorder that is extremely common in children and typically produces intermittent periods of hearing loss . More geTwenty-one ferrets were used in this study, of which ten were raised with an earplug while the remainder were raised normally. The left ear of the juvenile-plugged ferrets was first fitted with an earplug (EAR Classic) between postnatal day 25 (P25) and 29, shortly after the age of hearing onset in this species , and wasThe animals were trained from ∼P150 onward to approach the location of 200 ms broadband noise bursts with either flattened or randomized spectra in order to receive a water reward. Stimuli were presented from one of 12 speakers positioned in the horizontal plane. Reverse correlation maps were constructed from behavioral responses to randomized-spectrum stimuli. A simulated version of this task was also used to assess the performance of monaural and binaural models of sound localization see .R) with the sum of the MI values obtained using the other available spatial cues (MIother):Recordings were made under medetomidine/ketamine anesthesia from A1 units in response to VAS stimuli generated from acoustical measurements in each animal . These s


The purpose of this study is to identify whether or not different materials of softball bats  are a potential risk harm to hearing when batting players strike a 12′′ core .40 softball during slow, underhand pitch typical of recreational games. Peak sound pressure level measurements and spectral analyses were conducted for three controlled softball pitches to a batting participant using each of the different bat materials in an unused outdoor playing field with regulation distances between the pitcher's mound and batter's box. The results revealed that highest recorded peak sound pressure level was recorded from the aluminum (124.6 dBC) bat followed by the composite (121.2 dBC) and wooden (120.0 dBC) bats. Spectral analysis revealed composite and wooden bats with similar broadly distributed amplitude-frequency response. The aluminum bat also produced a broadly distributed amplitude-frequency response, but there were also two very distinct peaks at around 1700 Hz and 2260 Hz above the noise floor that produced its ringing (or ping) sound after being struck. Impulse (transient) sounds less than 140 dBC may permit multiple exposures, and softball bats used in a recreational slow pitch may pose little to no risk to hearing. Dangerous noise levels in a sporting event such as football (soccer), basketball, or baseball could come from crowd noise, referee whistles, and sporting equipment. Stadium or indoor area employees who work routinely in these environments can also be at risk of loud noise exposure –3. NatioDuring any sporting event, fans can increase noise levels by screaming, banging on the seats or bleachers, and, where permitted, using devices such as thundersticks and vuvuzelas. Vuvuzelas are trumpet-like instruments capable of producing sound pressure levels between 125 and 130 dB and those who blow these instruments can have significant distortion-product otoacoustic emission reductions that may lead to hearing loss [Sports officials (referees) who use whistles may be contributing to their own hearing loss and other auditory symptoms such as tinnitus . MoreoveOf recent interest and relevance to the present study, a study of modern golf drivers was conducted to determine peak levels and potential risk for hearing loss . This paThe foregoing discussion of sport-related noise exposure from crowds, officiating equipment, or sporting equipment is pervasive. However, we were not aware of studies formally evaluating peak sound pressure levels of softball bats. It is generally well known that aluminum bats produce a characteristic “ping” sound, while wooden bats produce more of a “crack” sound. Often times, the “ping” is perceived much louder, dampens less quickly, and is heard at further distances. For these reasons, the present study was designed to measure peak sound levels of three different softball bat materials  with balls thrown using a recreational slow pitch. This study bears relevance to audiologists and otologists who may encounter patients with noise-induced hearing loss or other auditory symptoms  due to sporting equipment. When hearing is unprotected from high levels of noise, whether the noise has a continuous or impulse quality, individuals may present with hearing loss (or other auditory processing problems ), tinnit In this study, a batter participant was tasked to hit softballs using three different softball bats delivered by a pitcher participant, each with amateur and collegiate softball experience. The same two participants were available on two measurement days. All procedures received prior approval by the Human Subjects Review Board at the University of Arkansas at Little Rock (Protocol number 13-058).Each of the three softball bats had a weight of 26.5 ounces and length of 32 inches. All were manufactured by Worth . The specific models used were Storm , Mayhem (composite), and Mayhem Ash (wood). Unlike wood and aluminum bats, composite bats are the latest technology and can be made out of graphite-fiber composite or have an aluminum core with a graphite lining. Core .40 softballs were used in this study. A core .40 softball is considered a low core and does not have as much “bounce” as a core .44 or .47. All measurements were performed at an empty community softball complex. Sound measures were only taken on days when the temperature was above 65°F (18.3°C) as temperatures is less than 60°F (15.5°C) degrees can result in damage to the bats.A sound level meter with oscilloscope setup was used to capture time domain waveforms and also record sound level measurements. The setup included a PC-based laptop with PicoScope software and USB-based PicoScope oscilloscope  plugged into the laptop and output of the Brüel & Kjær Type 2250  sound level meter coupled to the PicoScope. A Type 4189 1/2′′ microphone was used to capture the levels of the bat striking the softball. The sound level meter was calibrated before all sound measurements, and all recorded peak levels were measured using a C-weighted dB filter. Although the A-weighted measurements are most commonly reported, C-weighted peak sound levels of 140 dB for impulse-type noise are also often reported as the level that should not be exceeded by some countries and independent organizations . The parThe sound level meter was placed in the opposite batter's box at a spatial position as close to the height and distance of the batter's left ear as possible. To obtain reasonable position, the batter was asked to swing the bat while the height and distance of the microphone were relative to the home plate. This microphone positioning should give the most accurate representation of the effects of the impulse noise of a bat hitting a ball on an individual's hearing, minus any head-torso baffle effect. Slow underhand pitches were delivered from the pitcher's mound at regulation distance for softball, which is 45 feet (13.7 meters). Three controlled pitches were delivered for each material of bat. An average for each scenario was taken after 3 pitches for each bat on each day. An overall average was later taken for each bat type over the two days of measurements.M = 115.9), composite = 114.1–121.2 dBC (M = 117.8), and aluminum = 120.2–124.6 dBC (M = 122.6) (see The peak levels ranged as follows: wood = 113.1–120.0 dBC (2.6) see . As the All impulse sounds were no more than 0.111 ms in duration and were submitted to spectral analysis. The composite and wooden bats had a smooth, broad spread of energy and were similar to one another. On the other hand, the aluminum bat produced a spectrum, also broad, but there were clear areas of multiple peaks of energy above the noise floor. Two very distinct peaks emerged from the noise floor around 1700 Hz and 2260 Hz, which coincides with the “ping” of the aluminum bat. Representative time domain waveforms as well as the spectrum for the highest measured sound levels for each bat are shown in The present study investigated the peak sound pressure levels, time duration, and spectra of three different softball bat materials striking a .40 core softball for potential threat to human hearing. None of the levels recorded met or exceeded the 140 dBC ceiling limit for allowable exposure . HoweverBatting practice facilities are known to have a high reverberant acoustical quality. They usually have concrete flooring and are in sheet metal or exposed concrete block buildings. This environment can become very loud over time and increasingly louder with larger groups of batters hitting during the same practice session. In a practice type setting, a typical batter could easily hit 100 to 150 balls in one session. Moreover, a single batting session may take about 1 or 2 hours and maximum allowable doses may be reached faster than expected.The present study can only be generalized to recreational softball with slow underhand pitch. It is assumed that peak sound pressure levels are higher with faster underhand softball pitches and faster overhand baseball pitches. While we used a live pitcher with a controlled slow, underhand pitch, future research on other pitch speeds could be explored as well as the use of a pitching machine. In summary, slow underhand softball pitches most likely pose little to no risk to hearing, but batting practice with multiple impulse sound exposures could put an individual at risk of temporary to permanent threshold shift with bat materials that produce high intensity sounds. Hearing protection during long batting practice sessions may be recommended.


C1A cysteine peptidases are synthesized as inactive proenzymes. Activation takes place by proteolysis cleaving off the inhibitory propeptide. The inhibitory capacity of propeptides from barley cathepsin L and B-like peptidases towards commercial and barley cathepsins has been characterized. Differences in selectivity have been found for propeptides from L-cathepsins against their cognate and non cognate enzymes. Besides, the propeptide from barley cathepsin B was not able to inhibit bovine cathepsin B. Modelling of their three-dimensional structures suggests that most propeptide inhibitory properties can be explained from the interaction between the propeptide and the mature cathepsin structures. Their potential use as biotechnological tools is discussed. Plant proteolysis is a complex process that involves broad metabolic networks, different sub-cellular compartments and various types of peptidases, mainly cysteine-, serine-, aspartic- and metallo-peptidases From crystal structures of procathepsins B and L from mammals To become active, the C1A peptidases need to be processed either autocatalytically or with the aid of processing enzymes Selectivity of propeptide inhibition is a crucial feature to be addressed for using propeptides as biotechnological tools. In this way, many mammalian C1A propeptides not only are able to inhibit their cognate enzymes, but have the capacity to inhibit in trans several but not all related peptidases E. coli cultures. Bands detected after SDS-PAGE were in accordance with the expected molecular weights of purified propeptides, which rank from 12 kD of HvPap-19pro to 19 kD of HvPap-16pro  against the C1A cysteine peptidases were determined and showed different target specificities  was able to inhibit the activity of the cathepsin L-like peptidases HvPap-4 and HvPap-16. However, it did not inhibit the activity of the bovine cathepsin B, which was also not inhibited by any of the cathepsin L-like propeptides tested.To fully characterize the inhibitory properties of C1A propeptides in barley, we selected four cathepsin L-like  peptidases and one cathepsin B-like (HvPap-19) that had been previously described ap-16pro . In vitrs tested . The inhficities . OverallTo explain the lack of inhibition of HvPap-19 propeptide on bovine cathepsin B activity, we modelled the structure of both proteins using the crystallographic structure of the human procathepsin B as a template. Bovine cathepsin B and barley cathepsin B HvPap-19 aligned to human procathepsin B at sequence identities of 84.8% and 43.1%, and with Q-MEAN Z-scores of -1.55 and -3.81, respectively. Q-MEAN Z-score is a useful measure for the description of the absolute quality of theoretical models and is a valuable measure for identifying significant errors. Q-MEAN Z-scores less than -4.0 indicate that any part of the protein structure is not modelled correctly. These results imply a very accurate model for bovine cathepsin B and a relatively accurate model for barley HvPap-19. Major differences may be assumed from the models . The occTo explain the differences of inhibition of barley propeptides on barley cathepsin L-like activities, the structures of the HvPap-4, -6, -10, and -16 proteins were modelled using the crystallographic structure of the papaya cathepsin L-like procaricain as a template . Barley Ki values should be explained by differences in the interaction of some key amino acid residues. An example of how spatial changes in the orientation of the side chain of one amino acid can hinder the interaction propeptide-peptidase in presented  were inhibited by their cognate propeptides Acanthoscelides obtectusHeterodera glycines (plant nematode pest) reduced soybean cyst nematode infection Cysteine peptidases propeptides have been suggested to be potent inhibitors with the ability to control animal parasites and plant pests. Thus, C1A cysteine peptidases of Propeptides of C1A cysteine peptidases have been shown as tight-binding inhibitors of their cognate enzymes, but also of related peptidases As recombinant cathepsins B from plants have not been purified yet, the interspecific inhibitory effects of propeptides on commercial bovine cathepsin B was tested. The occluding loop of cathepsin B has been shown to prevent the propeptide of L-like cathepsins from binding the active site For cathepsin L-like peptidases, we used the barley members previously purified and characterized In conclusion, selectivity of interaction between plant C1A cysteine peptidases and propeptides becomes a complex feature. Molecular modelling of three-dimensional protein structures has become a powerful tool to explain in broad sense the specificity in the interactions. However, as side chain packing is the most difficult part of comparative modelling, further assays should be done to fully understand propeptide/peptidase interactions in order to use plant propeptides as regulators of C1A cysteine peptidases in biotechnological systems.E. coli BL21 CodonPlus (Stratagene). Bacterial cells were grown at 37°C to an OD550 of ca. 0.5 and induced with 1 mM IPTG  for 2 h 30 min, harvested and processed. The fusion proteins with the histidine tail were purified using a His-Bind Resin (Novagen) following the manufacturer's instructions. Purification process was checked by SDS-PAGE. The final protein concentration was quantified by the BioRad kit with bovine serum albumin (BSA) as standard. Additional MALDI-TOF analysis was performed to check molecular mass and propeptide identity by peptide mass fingerprint after trypsin digestion.The cDNA fragments spanning the putative propeptide sequences  were derived from HvPap-4, -6, -10, and -17 barley genes E. coli cultures as described E. coli transformed with the empty expression vector were used. Enzyme concentrations were determined by active-site titration with the irreversible inhibitor L-trans-Epoxysuccinyl-leucylamido(4-guanidino)butane (E-64). Similarly, the concentration of correctly folded propeptides was determined by titration with different barley papain-like peptidases previously titrated with E-64. The kind of inhibition was determined from Lineweaver-Burk plots (1/V versus 1/[S]), and confirmed by lineal correspondence between IC50 and [E] values and IC50 and [S] values Ki [Ki(app)] values were calculated by non-linear regression using the GraFit program Ki) were then calculated according to the equation Ki = Ki(app)/(1+[S]/Km) using Km values calculated by non-linear regression of the Michaelis-Menten equation using the GraFit program.Recombinant barley HvPap-4, -6, -10, -16 cysteine peptidases were purified and activated from The amino acid sequences of barley C1A cysteine peptidases were extracted from the NCBI GenBank. BlastP searches for cathepsin B-like cysteine peptidases were made using the amino acid sequence of the HvPap-19 protein The three-dimensional structures of the barley cysteine peptidases and the bovine cathepsin B were modelled using the standard automated routine of SWISS-MODEL program Figure S1Comparison of the amino acid sequences of the cathepsin B-like cysteine proteases. The alignment was generated using the MUSCLE program. Pp, Physcomitrella patens; Sm, Selaginella moellendorffii; Os, Oryza sativa; Hv, Hordeum vulgare; Pt, Populus trichocarpa; At, Arabidopsis thaliana.(DOC)Click here for additional data file.Table S1Information about the cathepsin B and L-like proteins used in the alignments.(DOC)Click here for additional data file.


AbstractMycetophagidae (hairy fungus beetles) of the Maritime Provinces of Canada are surveyed. Seven species in the genera Mycetophagus, Litargus, and Typhaea are found in the region. Six new provincial records are reported including Mycetophagus punctatus and Mycetophagus flexuosus, whichare newly recorded in the Maritime Provinces. The distribution of all species is mapped, colour habitus photographs of all species are figured, and an identification key to species is provided. The discussion notes that four of the species found in the region are apparently rare, possibly due to the history of forest management practices in the region; a situation similar to that of a significant proportion of other saproxylic beetles found in the Maritime Provinces.The  Mycetophagidae (hairy fungus beetles) are a family of relatively small, fungus-eating beetles. Only five genera and 26 species are known in North America, 15 of which have been recorded in Canada (Mycetophagus quadriguttatus Müller and Typhaea stercorea (Linnaeus), have previously PageBreakbeen recorded from the Maritime Provinces of Canada  , an adventive Palaearctic species, is associated with a large variety of moldy substances and is found both outdoors in natural environments, and indoors in association with a variety of stored products  . Speciesehydrate . Typhaeaproducts . The bioing logs . The preAcronyms JCCJeffrey Ogden Collection, Truro, Nova Scotia, CanadaJOCKent Island Collection, Bowdoin College, Brunswick, Maine, USAKICNova Scotia Agricultural College, Bible Hill, Nova Scotia, CanadaNSAC	Nova Scotia Museum, Halifax, Nova Scotia, CanadaNSMCNova Scotia Department of Natural Resources Insectary, Shubenacadie, Nova Scotia, CanadaNSNRRichard Migneault Collection, Edmundson, New Brunswick, CanadaRMC, flight intercept trap.FITAbbreviations: PageBreakAn identification key to species [adapted from Mycetophagus species are variable.* Note: elytral markings on Mycetophagidae were examined – 8 from New Brunswick, 149 from Nova Scotia, and 18 from Prince Edward Island. Included were specimens of seven species in three genera. Mycetophagus flexuosus Say is newly recorded in the Maritime Provinces from New Brunswick; Mycetophagus punctatus Say is newly recorded in the Maritime Provinces from Nova Scotia; Mycetophagus serrulatus Casey is newly recorded in New Brunswick; Mycetophagus pluripunctatus LeConte is newly recorded in New Brunswick; Mycetophagus quadriguttatus Müller is newly recorded in Nova Scotia; and Litargus tetraspilotus LeConte is newly recorded in Prince Edward Island – a total of five new provincial records, two of which are newly recorded PageBreakin the region. Four species are known from New Brunswick, six from Nova Scotia, and two from Prince Edward Island ; Edmundston, 47°22.285'N; 68°14.663'W, 22 August 2010, R. Migneault, in polypore on dead aspen log .Mycetophagus flexuosus is newly recorded in the Maritime Provinces from New Brunswick (Pleurotus ostreatus) Fries; Tramates versicolor (Fr.) Pil.); and Piptoporus betulinus) (Fr.) Kar.runswick . Cline aSay, 1826NOVA SCOTIA: Halifax Co.: Soldier Lake, 7 June 2005, J. Ogden, spruce beetle trap ; Hants Co.: Smileys Park, 6 July 2005, J. Ogden, spruce beetle trap .Mycetophagus punctatus Say is newly recorded in the Maritime Provinces from Nova Scotia. Both specimens were collected in the central mainland of Nova Scotia (Quercus velutina Lamb.) in Virginia (Polyporus radicatus Schw.) in Iowa (Pleurotus ostreatus) (Piptoporus betulinus) growing on gray birch  in New York  ; and on New York .Casey, 1900NEW BRUNSWICK: Charlotte Co.: Grand Manan archipelago, Kent Island, 23 July 2012, M. Steck, balsam fir forest, sweeping . NOVA SCOTIA: Annapolis Co.: Durland Lake, 21 June 2003, P. Dollin, hemlock/balsam fir/black spruce forest (120+ years), bracket fungi on white birch .Mycetophagus serrulatus Casey is newly recorded in New Brunswick. The species was reported from Nova Scotia by Betula papyrifera Marshall). Pleurotus ostreatus).PageBreakLeConte, 1856NEW BRUNSWICK: Madawaska County: Edmundston, 47°22.285'N; 68°14.663'W, 22 August 2010, R. Migneault, in polypore on dead aspen log . NOVA SCOTIA: Antigonish Co.: Cape George Point, 23 June1993, M. LeBlanc, funnel trap ; Colchester Co.: Kemptown, 1 June 1995, 28 June 1995, C. Corkum, young deciduous forest, FIT ; Upper Bass River, 18 May 1995, C. Corkum, old deciduous forest, FIT ; Upper Bass River, 3 June 1995, C. Corkum, old deciduous forest, FIT ; Cumberland Co.: East Leicester, 2 June 1995, C. Corkum, old deciduous forest, FIT ; East Leicester, 14 June 1995, C. Corkum, old deciduous forest, FIT ; East Leicester, 15 June 1995, C. Corkum, old deciduous forest, FIT ; Fox River, PageBreak17 May 1995, C. Corkum, young deciduous forest, FIT ; Fox River, 3 June 1995, C. Corkum, young deciduous forest, FIT ; Harrington River, 13 July 1995, C. Corkum, young deciduous forest, FIT ; Wentworth, 21 May-5 July 1965, B. Wright, sugar maple forest, window trap ; Halifax Co.: Halifax, 1 December 1986, B. Wright ; Soldier Lake, 30 July 2004, D. MacDonald, spruce beetle trap ; Lunenburg Co.: Card Lake, 2-15 June, 1997, D.J. Bishop, red spruce/hemlock forest (old growth), FIT ; Yarmouth Co.: Wellington, 23-29 August 1992, J. & F. Cook, mixed forest .Mycetophagus pluripunctatus LeConte is newly recorded in New Brunswick. The species was reported from Nova Scotia by Piptoporus betulinus), Pleurotus ostreatus), and Spongipellis unicolor (Schw.) growing on a fallen white oak  in Arkansas. Schwartz (1876) said it was “abundant in fungus” in Michigan.PageBreakMüller, 1821NOVA SCOTIA: Annapolis Co.: Paradise, 11 June 2005, K. Webster, spruce beetle trap ; Colchester Co.: Balmoral Mills, 19 June 1974, B. Wright, grist mill ; Kings Co.: Kentville, 10 August 2005, D.H. Webster, compost heap, moldy corncobs .Mycetophagus quadriguttatus Müller is newly recorded in Nova Scotia NEW BRUNSWICK: Northumberland Co.: Tabusintac, 13 June 1939, 26 July 1939, W.J. Brown . NOVA SCOTIA: Colchester Co.: Truro, 4 March 1919, collector not recorded . PRINCE EDWARD ISLAND: Prince Co.: Central Bedeque, 29 July 1954, F.M. Cannon .Eighty-two specimens  were examined. The earliest records from each province are: Typhaea stercorea (Linnaeus) was reported from New Brunswick, Nova Scotia, and Prince Edward Island by Typhaea stercorea has been found in corn fields (on decaying kernels of exposed ears), warehouses, stores, flour mills, mangers, railway boxcars, dwellings, and granaries in stored grain and seeds, tobacco, peanuts, cacao, corn, millet, wheat, apricots, and moldy grape skins, as well as in nests of swans and moorhens  South and Central America (The dates of earliest detection are given above: New Brunswick (1939), Nova Scotia (1919), and Prince Edward Island (1954). ontinent , and is  America .LeConte, 1856NOVA SCOTIA: Cape Breton Co.: East Bay, 9 September 2003, C.W. D’Orsay ; Colchester Co.: Bible Hill, 8 July 2004, K.R. Aikens, pasture, sweep ; Bible Hill, 14 June 2005, S.M. Townsend, sweep ; Debert, 9 June 1994, J. Ogden ; Masstown, 7 September 2002, C.G. Majka, marshy swamp ; Shubenacadie, 26 August 1997, J. Ogden ; Digby Co.: Brier Island, Pond Cove, 9 August 2004, J. Ogden & K. Goodwin, knapweed, sweep ; Brier Island, Pond Cove, 10 August 2004, J. Ogden & K. Goodwin, sweep ; Brier Island, Westport, 9 August 2004, J. Ogden & K. Goodwin, grassland, sweep ; Halifax Co.: Big Indian Lake, 16 July 2003, P. Dollin, Picea rubens forest (80-120 years), in rotting mushroom ; Point Pleasant Park, 15 August 2000, 7 September 2000, C.G. Majka, mixed forest ; Point Pleasant Park, 9 September 2000, 2 June 2002, 23 July 2002, C.G. Majka, coniferous forest ; Point Pleasant Park, 12 May 2001, 10 June 2001, 25 May 2002, C.G. Majka, coniferous forest, on Picea rubens ; Point Pleasant Park, 19 May 2001, 29 May 2001, C.G. Majka, coniferous forest, on Pinus strobus ; 29 July 2001, 18 August 2001, Point Pleasant Park, C.G. Majka, mixed forest ; PageBreakPoint Pleasant Park, 9 May 2002, C.G. Majka, coniferous forest, on Abies balsamea ; Point Pleasant Park, 9 June 2002, C.G. Majka, mixed forest, on Aralia hispida ; Point Pleasant Park, 7 July 2002, C.G. Majka, seashore ; Point Pleasant Park, 14 September 2002, C.G. Majka, marsh, on herbaceous vegetation ; Point Pleasant Park, 30 June 2004, C.G. Majka, coniferous forest, on Pinus sylvestris ; West Dover, 7 September 2003, C.G. Majka, coastal barrens, heaths ; Kings Co.: Aldershot, 5 August 1949, 2 August 1949, 10 August 1949, 20 August 1949, 16 May 1950, H.T. Stultz ; Greenwich, 29 May 1958, H.T. Stultz ; Kingston, 30 June 2002, C.G. Majka, sandy pine barren ; Queens Co.: Eight Mile Lake, 11 August 2003, P. Dollin, PageBreakPicea rubens forest (40-80 years), in vegetation, sweep ; Little Ponhook Lake, 1 August 1993, B. Wright, in oak apple galls ; Ponhook Lake nr. Greenfield, 13 July 1993, J. Cook, ultraviolet light trap ; Shelburne Co.: Clyde River Road, 16 July 1992, S. & J. Peck, forest, car net ; Forbes Point, 9 July 2007, R. Gorham, grass/alders ; Victoria Co.: Cape Breton Highlands: Kelly Rd, 24 June 2005, J. Ogden, malaise trap ; Yarmouth Co.: Moses Lake, 8 km N of Argyle, 17-22 July 1993, J. & T. Cook, mixed forest, FIT . PRINCE EDWARD ISLAND: Queens Co.: Cavendish, 19 July 2001, C.G. Majka, coastal vegetation ; Princeton-Wharburton Road, 19 August 2002, C.G. Majka, old field ; St. Patricks, 18 August 2002, C.G. Majka, old field ; St. Patricks, 29 June 2003, C.G. Majka, mixed forest .Litargus tetraspilotus LeConte is newly recorded from Prince Edward Island. PageBreakmany records from the southern mainland of Nova Scotia, Cape Breton Island, and Prince Edward Island , jack pine (Pinus sylvestris L.), red spruce (Picea rubens Sarg.), balsam fir  Mill.), on deciduous, and herbaceous vegetation, on bristly sarsaparilla , and in a rotting mushroom. Litargus tetraspilotus as an inquline inhabitant of oak apple galls on red oak (Quercus rubra L.) induced by Andricus  sp. (Cynipidae) wasps. Phoradendron flavescens (Pursh) Nutt., and Ulyschen and Hanula (2010) reared it from decomposing loblolly pine (Pinus taeda L.) logs in South Carolina.In the Maritime Provinces Typhaea stercorea and Litargus tetraspilotus are abundant and widely distributed in the Maritime Provinces. Mycetophagus pluripunctatus appears to be uncommon but widely distributed on the mainland of Nova Scotia. The other four species of mycetophagids – Mycetophagus punctatus, Mycetophagus flexuosus, Mycetophagus serrulatus, and Mycetophagus quadriguttatus – are all represented by a handful of specimens or less. They would all appear to qualify asPageBreak “apparently rare” saproxylic beetles as defined by Endomychidae and Erotylidae of the Maritime Provinces, two other families of beetles closely associated with fungi, Mycetophagus, three of which are closely associated with saproxylic fungi, may belong to this same suite of insects for similar reasons.In general, mycetophagids have received rather little attention by researchers in North America, and the bionomics of many species have not been carefully investigated. Certainly this is true in the Maritime Provinces and additional fieldwork in the region is required to ascertain more about their distribution, abundance, bionomics, and ecological role in the habitats that they inhabit.


Tinnitus is proposed to be caused by decreased central input from the cochlea, followed by increased spontaneous and evoked subcortical activity that is interpreted as compensation for increased responsiveness of central auditory circuits. We compared equally noise exposed rats separated into groups with and without tinnitus for differences in brain responsiveness relative to the degree of deafferentation in the periphery. We analyzed (1) the number of CtBP2/RIBEYE-positive particles in ribbon synapses of the inner hair cell (IHC) as a measure for deafferentation; (2) the fine structure of the amplitudes of auditory brainstem responses (ABR) reflecting differences in sound responses following decreased auditory nerve activity and (3) the expression of the activity-regulated gene Arc in the auditory cortex (AC) to identify long-lasting central activity following sensory deprivation. Following moderate trauma, 30% of animals exhibited tinnitus, similar to the tinnitus prevalence among hearing impaired humans. Although both tinnitus and no-tinnitus animals exhibited a reduced ABR wave I amplitude (generated by primary auditory nerve fibers), IHCs ribbon loss and high-frequency hearing impairment was more severe in tinnitus animals, associated with significantly reduced amplitudes of the more centrally generated wave IV and V and less intense staining of Arc mRNA and protein in the AC. The observed severe IHCs ribbon loss, the minimal restoration of ABR wave size, and reduced cortical Arc expression suggest that tinnitus is linked to a failure to adapt central circuits to reduced cochlear input. Tinnitus is a brain disorder causally linked to noise-induced hearing loss, cochlear damage To study central responsiveness to auditory trauma related to tinnitus, we used a tinnitus animal model that was designed to minimize stress based on access to sugar water as a positive reward Arc expression is involved in acute and long-lasting alterations of network activity as a consequence of altered sensory input In order to identify a tinnitus-specific difference between equally acoustically exposed animals with and without tinnitus (tinnitus-trait), we exposed rats binaurally to noise and behaviorally identified animals with tinnitus. In this model, rats are trained to associate white noise with a sugar water reward and silence with no reward To increase the likelihood of detecting a tinnitus-trait, we used various noise conditions. To assure specificity, tinnitus was evaluated in both groups by the same criteria (silence activity >0.1), independently of the hearing loss of individuals. One group of animals was exposed to noise at a sound pressure level of 120 decibels  at 10 kilo Hertz (kHz) for 1 hour (h) and analyzed 6 days (d) post exposure, when no further recovery of hearing was expected to occur. Another group of animals was exposed to 120 dB SPL for 1.5 h. Since the exposure for 1.5 h resulted in a stronger trauma, we analyzed these animals 30 d post exposure to make certain that the hearing and the tinnitus had recovered at its best and no further recovery of threshold was expected.Hearing function was studied by ABRs evoked with short acoustic stimuli before and after noise exposure, at the same day as behavioral testing. ABRs represent the summed activity of neurons in the ascending auditory pathway and are measured by averaging the evoked electrical response recorded via subcutaneous electrodes.Animal care, procedures, and treatments were performed in accordance with institutional and national guidelines following approval by the University of Tübingen, Veterinary Care Unit, and the Animal Care and Ethics Committee of the regional board of the Federal State Government of Baden-Württemberg, Germany .Adult female Wistar rats weighing 200–300 g were exposed in anesthesia to sound (120 dB SPL), or sham exposed with loud speaker switched off, for either 1 h or 1.5 h, at 10 kHz and sacrificed 6 d, or 30 d after Animals were anesthetized with a mixture of ketamine–hydrochloride  and xylazine hydrochloride , injected i.p. Anesthetized rats were binaurally exposed to free field inside a reverberating chamber . To eliminate furthermore the effects of any possible inhomogeneity in the acoustic field and to avoid that ear were positioned at sites of pressure wave extinction animals were slowly constantly moved through the sound field by means of a rotating turntable and were repositioned on this table in intervals of 30 minutes to ensure a homogeneous exposure of all animals on both ears. The reverberating chamber was equipped with top and side wall mounted speakers . The loudness and spectrum of the traumatizing sound was constantly monitored my means of a microphone placed in the centre of the chamber and customized computer software analyzing the frequency spectrum by fast fourier transformation (FFT). The traumatizing sound had a narrow peak at 10 kHz and sidebands at 20 kHz (−40 dB) and 30 kHz (−60 dB). Additional side bands were below 50 dB SPL and therefore did not exceed the normal laboratory background noise.Additional doses of anaesthetics were administered if needed and body temperature maintained by heating pads and lamps.ABRs were measured in a soundproof chamber  as described For stimulus generation and recording of ABRs, a multi-function IO-Card  was used, housed in an IBM compatible computer. Sound pressure level was controlled with an attenuator and amplifier (Wulf Elektronik). Filter settings, ABR spectral components (by FFT), potential stimulus artefacts were examined beforehand to ensure that ABR signal waveform, amplitude and latencies of distinct peaks in the ABR were not affected by the chosen hardware and software parameters. In particular, to reduce physical stress of the animals by long lasting anaesthesia to a minimum, ABR measurement times were reduced to a minimum by increasing stimulus repetition rates to 80/s, minimizing repetition numbers for clearly suprathreshold signals (when ABR wave amplitudes were exceeding ±4 µV), and reducing sample rates to 10 kHz to reduced delay times by computer online analyses. In control studies it was validated that the protocols that were finally used , gave homogeneous results as compared to higher repetition numbers (512) and higher (100 kHz) sample rates.Hearing threshold was determined by the lowest sound pressure that produced visually distinct evoked potentials from above threshold to near threshold.waveform analysis), and the peak input-output function (peak I/O) was analyzed.For each individual ear, the auditory brainstem response (ABR) waveform to click stimuli  no-tinnitus rats before exposure, (2) no-tinnitus rats after exposure, (3) tinnitus rats before exposure, and (4) tinnitus rats after exposure. For all four groups, the ABR functions were analyzed at a stimulus level of 90 dB SPL (a high stimulus level) and at 40 dB above hearing threshold (40 dB hearing level). The average waveforms gave a qualitative and temporal assessment of changes in amplitude ranges in the ABR function.The ABR wave data for the click stimuli were analyzed for peak and through amplitudes and the latencies by customized computer programs.From individual ABR waves to click stimuli, peak amplitudes and peak latencies were collected, grouped in clusters of similar peak amplitude and latencies, and averaged for ABR wave input-output (I/O) analysis. Wave amplitudes were defined as peak to peak amplitude of a negative peak (n) followed by a positive (p) peak. Clusters of peaks were found at average latencies n0.9-p1.2, n1.3-p1.6, n1.9-p2.4 or p2.7, n3.6-p4.9, and n7.1-p9.4. The ABR recordings are presented for the first 10 ms recording time. To affirm that the peak at p9.4 was not missed out due to a peak latency longer than 10 ms, the whole recording cycle consisting of a compression and a rarefaction stimulus  was analyzed and the peak position approved within the first 10 ms. For selected peaks and troughs the I/O functions were derived from the peak-to-peak amplitudes at all recorded stimulus sound pressure levels. Three peak classes were selected: (1) early peaks ; (2) delayed peaks (at 3.6–4.9 ms), found in the range of the greatest loss of the ABR waveform ; and (3) late peaks (at 7.1–9.4 ms), as these peaks fall into the time range of thalamic activation.Pre: the amplitudes at point 1 to 100 (0–10 ms) before exposureAPost: the amplitudes at point 1 to 100 (0–10 ms) after exposureVarPre variance of the click-ABR wave before exposureCorF: correlation factor (covariance/variance) The similarity in the waveform of the ABR before and after an acoustic noise exposure is founded on changes of all peak amplitudes and all peak latencies. In human tinnitus studies, often the relation of wave-I to wave-V is reported The CorF therefore provides a normalization of the covariance with respect to the variance of the measurement before exposure. In contrast to the correlation by the Pearson Momentum Coefficient that is not sensitive to overall amplitude changes, the CorF reflects changes in waveform and amplitude. Typical values are 1.0 for optimal correlation between waveforms of similar amplitude values. Loss of similarity and loss of amplitude typically result in CorF values approaching null.Within 3–4 months before the noise exposure, rats were trained using operant conditioning to perform a specific motor task  when perceiving an external sound cue (presented for 1–3 minutes) and to cease this motor activity during periods of silence . A conditioning session lasted 20–60 minutes containing 10–30 silence and sound periods, sound stimuli were varied between 70 and 10 dB SPL (in steps of 10 dB), and 32–38 sessions were required to train the rats to meet the training criterion. Correct indication of tinnitus of either rats was tested by transient tinnitus induction after salicylate injection (350 mg/kg bodyweight) Cochlear and brain tissues were dissected as previously described Rat cochleae were isolated, fixed, cryosectioned, and stained as described in Riboprobes were designed as described in Correlation factors and hearing loss were compared using Student’s t-Test. The statistical significance at the alpha level of 0.05 is indicated in the figure legends.Tinnitus behavior was compared using the Mann-Whitney-U Test. For the ribbon counts of apical, medial and basal cochlear turns, P-values were corrected for alpha-shift by multiple testing using the Bonferroni-Holms procedure.Ribbon numbers from 3–5 animals from 3 independent experiments per group were counted and compared by 1-way ANOVA. For the ribbon counts of apical, medial and basal cochlear turns, P-values were corrected for alpha-shift by multiple testing using the Bonferroni-Holms procedure.t test, with alpha = 0.05. Cells were counted in 3 animals per group in three independent experiments.Arc mRNA expressing cells were counted in the AC as previously described Behavioral testing ABR thresholds for click-stimuli  and freqAlthough the various intensities of sound exposure led to a variable amount of hearing loss, the 1 h exposed tinnitus rats and the 1.5 h exposed tinnitus-free rats had a similar low frequency hearing function , though We counted IHC ribbons from 3–5 animals per group  as a correlate of the number of afferent auditory fibers These data indicate a severe damage of the IHC synapse in high-frequency cochlear turns of animals with tinnitus in comparison to tinnitus-free animals with similar hearing thresholds for click auditory stimuli .IHC ribbons determine the generation of spikes in afferent auditory fibers Click-evoked ABR waveform amplitudes before and after noise exposure were compared for differences in signal amplitudes for 1 h  and 1.5 h exposure duration  at 40 dB above threshold and 90 dB SPL . These cAs expected from the permanent, although mild, hearing loss to click stimuli within both groups , a reducThis suggests that both, high-SR, low level and low-SR, high level auditory fibers might be affected. This was studied by the I/O growth of the dominant peaks (indicated in We need to point out that click stimuli are used that are dominated by frequencies lower than 10 kHz (see methods 2.2). As shown by Delayed and Late). This suggested that in tinnitus-free animals, responsiveness to sound at the level of the lateral lemniscus and inferior colliculus (IC) (Delayed) or at the level of the IC output and medial geniculate body (MGB) (Late) is enhanced, in comparison to animals with tinnitus. As cochlear CF regions of best hearing of the animals are the main source of ABR waves generated in higher brain regions  responsiveness of central circuits.The present study compared markers for deafferentation, brainstem responsiveness and homeostatic plasticity in equally acoustically exposed animals that were behaviorally separated into groups of animals with and without tinnitus. A characteristic pattern of severe IHCs ribbon loss, insufficiently restored late ABR wave and a failure to mobilize Arc in the cortex could be linked to high-frequency hearing impairment and tinnitus. This finding is discussed in the context of a failure to recruit central gain following cochlear deprivation as a correlate of tinnitus.To assure the specificity of a tinnitus-specific trait, this trait should be a discrete property, independent of hearing loss. The rats with tinnitus after a mild trauma (1 h) and the rats in the no-tinnitus group after mild (1 h) and a more extensive trauma (1.5 h) had a similar low frequency hearing. This indicates that the amount of threshold loss required for tinnitus induction is not necessarily of a uniform dimension, as already suggested by other authors Irrespective of the amount of hearing impairment, we observed that tinnitus was coupled to severe IHC ribbon loss after both exposure durations in 30% of animals, an incidence also observed among hearing-impaired humans Approximately 20 ribbons tether synaptic vesicles in the active zones of an IHC Using click stimuli spectrally dominated by frequencies below 10 kHz, we found mild and similar but significant hearing loss 3–18 dB,  and ABR The restored ABR waves observed here in hearing impaired animals without tinnitus mainly reflect spreading activity through the VCN but not DCN As our results show that ABR waves are restored in the tinnitus-free situation, the findings do not support the idea that increased responsiveness in CN target neurons is a correlate for tinnitus. The more restored ABR waves in tinnitus-free animals may reflect a form of loudness recruitment or hyperacusis and the less restored ABR waves in tinnitus animals a reduced capacity to compensate missing gain in the ascending pathway. Data would thereby experimentally support a recent computational model that suggests steeper rate level function in brainstem neurons as the source of non-linear gain that produces loudness recruitment and hyperacusis In the cortex, Arc mRNA is expressed in non-GABAergic glutamatergic neurons Arc mRNA is directly transported within minutes to distal dendrites following LTP like activity Since Arc protein is rapidly degraded While Arc mobilization in the cortex may elevate the sensitivity of cortical pyramidal neurons The rapid deafferentation In conclusion, the current findings provide a rationale for the altered responsiveness of central circuitries after different degrees of IHC synapse and auditory fiber damage. The observed severe decline of ribbon numbers, the changes in ABR wave amplitudes and the failure to mobilize Arc in the AC strongly support the idea that increased neuronal activity in the auditory periphery is a compensation of peripheral input – avoiding tinnitus – rather than a correlate or even the origin of tinnitus.