Mycoplasma hyopneumoniae (M. hyopneumoniae) transmission and control. An effective gilt acclimation program should ideally reduce M. hyopneumoniae shedding at first farrowing, decreasing pre-weaning colonization prevalence and potential respiratory problems in fatteners. However, information on gilt acclimation practices is scarce in Europe. The aim of this study was to identify current acclimation strategies for M. hyopneumoniae in Europe using a questionnaire designed to assess 15 questions focused on gilt replacement status, acclimation strategies and methods used to ascertain its effect. A total of 321 questionnaires (representing 321 farms) were voluntarily completed by 108 veterinarians (from 18 European countries). From these farms, 280 out of 321 (87.2%) were aware of the health status of gilts on arrival. From these 280 farms, 161 (57.5%) introduced M. hyopneumoniae positive replacements. In addition, 249 out of 321 (77.6%) farms applied an acclimation process using different strategies, being M. hyopneumoniae vaccination  and the combination of vaccine and exposure to sows selected for slaughter  the most commonly used. Notwithstanding, only 53 out of 224 (23.6%) farms, knowing the M. hyopneumoniae initial status and performing acclimation strategies against it, verified the effect of the acclimation by ELISA , PCR  or both . This study showed that three fourths of the farms represented in this European survey have M. hyopneumoniae acclimation strategies for gilts, and one fifth of them verify to some extent the effect of the process. Taking into account that the assessment of acclimation efficacy could help in optimizing replacement gilt introduction into the breeding herd, it seems these practices for M. hyopneumoniae are still poorly developed in Europe.Gilts are considered to play a key role in  Mycoplasma hyopneumoniae (M. hyopneumoniae) is the etiological agent of swine enzootic pneumonia (EP), a respiratory disease that mainly affects growing and finishing pigs ), two (148 [46.3%]), three (51 [15.9%]), four (39 [12.2%]) and even five (2 [0.6%]) methods, respectively  farms, from which 161 (57.5%) were seropositive. Importantly, only 79 out these 280 (28.2%) farms confirmed the theoretical gilt status upon arrival. The most frequently used method to verify this status was ELISA . Results of such verification were not requested.Approximately half of the surveyed farms introduced external replacement gilts Table , whereasAge of replacement on arrival at sow farm (in case of farms with purchased replacement) or at internal selection site (in case of farms with own replacement) differed among studied farms, varying from 0 to 210 days Fig. . Gilt inM. hyopneumoniae acclimation strategies performed is shown in Table M. hyopneumoniae. From these 225 farms, 159 (70.7%) were located in Southern European countries and 66 (29.3%) in the rest of European participating countries. However, there were 24 farms that, although not having isolation units or not answering this question, performed acclimation process for M. hyopneumoniae (no more information was available regarding the specific site used to acclimate gilts). Additionally, from these 249 farms performing acclimation strategies, M. hyopneumoniae status of gilts on arrival was investigated in 224 farms .Information of M. hyopneumoniae was the most used acclimation procedure , followed by vaccination and animal exposure, with either culled sows  or pigs , and a combination of vaccination and both types of live animal exposure  (Table Vaccination against M. hyopneumoniae specific acclimation was performed by 76.4% (172/225) and 80.2% (77/96) of farms from Southern and rest of Europe, respectively. In both geographic regions vaccination was the most frequently used method, reaching values of around of 95%. Nevertheless, whereas in Southern regions combined vaccination with animal exposure (cull sows and/or pigs) was practiced, farms located in the rest of Europe mainly used vaccination alone.Finally, considering the farms where the initial status was known and the acclimation process was carried out, the verification of the effect of such strategies was performed in only 23.7% of the farms (53/224), being the combination of ELISA and PCR the most frequently used method. Importantly, the confirmation of theoretical gilt status upon arrival and after gilt acclimation to verify the effect was checked in 49.4% farms (39/79). Results of such verification were not requested.M. hyopneumoniae gilt status and acclimation practices conducted in European farms by means of a descriptive study. To date, no information about this issue is available in Europe and, in fact, minimal data do exist from around the world, with surveys performed in USA [The aim of the present study was to gain knowledge on d in USA  and Mexid in USA .M. hyopneumoniae in pig farms . Although the European situation might not be fully represented , no significant differences in terms of gilt acclimation practices were detected between the South of Europe  and the rest of participating countries (data not shown). Nevertheless, information obtained about M. hyopneumoniae current status on gilt acclimation should help depicting measures that can be potentially applied elsewhere. Additionally, the facts that all but one of the participants evaluated M. hyopneumoniae status of their farm and more than 80% were aware of their replacement status regarding this pathogen, suggest that EP is still a concern to swine industry. Notwithstanding, this assumption could be influenced by the fact that only concerned veterinarians on M. hyopneumoniae completed the questionnaire.Questionnaires were voluntarily responded, thus, they may have some inherent biases in the responses . Indeed,M.hyopneumoniae associated problems was based on presence of clinical signs accompanied with lung lesion scoring at slaughterhouse. Noteworthy, non-productive dry coughing and cranio-ventral pulmonary consolidation (CVPC), the usual clinical signs and lung lesions attributed to M. hyopneumoniae infection, can also be produced by other respiratory pathogens [M. hyopneumoniae involvement in clinical signs and lesions [M. hyopneumoniae status was evaluated based their assessment only on non-specific methods , suggesting that most European farms represented in this study performed an incomplete assessment of M. hyopneumoniae health status. This percentage is lower than Mexican survey (no data is available for US), since in that study, 71% of the respondents evaluated M. hyopneumoniae farms situation only according to clinical signs [Most of the questionnaire respondents reported that the assessment of athogens , 20, and lesions . A totalal signs .M. hyopneumoniae. Comparatively, percentage of positive replacement in the assessed European farms was similar  to that in the USA (55%), but lower than in Mexico (90%) [The introduction of external replacement into a swine herd is considered a potential risk of new pathogen introduction and farm health destabilization , 22, as co (90%) , 18.Interestingly, most farms (80.9%) had isolation facilities to acclimate, being the most utilized the AIAO system. In terms of type of management systems used to acclimatize gilts, a clear difference between participating European farms, USA and Mexico was observed. Whereas 75% and 72% of Mexican  and USA M. hyopneumoniae are not able to prevent bacterial colonization and the transmission between vaccinated pigs seems not to be significantly altered [M. hyopneumoniae shedders in 27.4% and 14.6% of studied farms, respectively; however their shedding status was not required in the questionnaire. These results are in agreement with previous studies in USA (34% of respondents utilized culled sows to acclimatize) and Mexico (27% of responders used culled sows and 10% piglets) [Replacement gilt acclimation methods used in Southern European farms were mainly based on vaccination alone or in combination with live animal exposure (culled sows or pigs), whereas farms from rest of participant countries utilized vaccination exclusively. These results were in line with the ones reported by USA and Mexican studies , 18, in  altered , 24–27. piglets) , 18. AccM. hyopneumoniae in seronegative farms or M. hyopneumoniae re-circulation/re-infection in seropositive ones.Finally, the relatively low percentage of farms verifying the acclimation process (23.7%) indicated that most of the surveyed farms did not evaluate gilt infection and shedding status at first farrowing. This situation coincided with the information reported by Mexican and USA studies, where only 20% and 14% of responders, respectively, validated the acclimation process. An inadequate acclimation process could imply that gilts would be a potential source of infection for its offspring and, therefore, leading to an outbreak of M. hyopneumoniae positive replacement stock, but only a minority assessed its health status on arrival. Likewise, most of participating farms performed a specific gilt acclimation procedure against M. hyopneumoniae. Moreover, the verification of this process was not a common practice.The present study shows that most of the European farms introduced


With the exception of primates, most vertebrates have laterally placed eyes. Binocular vision in vertebrates has been implicated in several functions, including depth perception, contrast discrimination, etc. However, the blind area in front of the head that is proximal to the binocular visual field is often neglected. This anterior blind area is important when discussing the evolution of binocular vision because its relative length is inversely correlated with the width of the binocular field. Therefore, species with wider binocular fields also have shorter anterior blind areas and objects along the mid-sagittal plane can be imaged at closer distances. Additionally, the anterior blind area is of functional significance for birds because the beak falls within this blind area. We tested for the first time some specific predictions about the functional role of the anterior blind area in birds controlling for phylogenetic effects. We used published data on visual field configuration in 40 species of birds and measured beak and skull parameters from museum specimens. We found that birds with proportionally longer beaks have longer anterior blind areas and thus narrower binocular fields. This result suggests that the anterior blind area and beak visibility do play a role in shaping binocular fields, and that binocular field width is not solely determined by the need for stereoscopic vision. In visually guided foragers, the ability to see the beak—and how much of the beak can be seen—varies predictably with foraging habits. For example, fish- and insect-eating specialists can see more of their own beak than birds eating immobile food can. But in non-visually guided foragers, there is no consistent relationship between the beak and anterior blind area. We discuss different strategies—wide binocular fields, large eye movements, and long beaks—that minimize the potential negative effects of the anterior blind area. Overall, we argue that there is more to avian binocularity than meets the eye. Tyto alba) and a few other birds of prey [Despite most vertebrates having their eyes positioned laterally rather than frontally, the discussion about binocular vision in vertebrates has traditionally revolved around the extraction of relative depth information from stereopsis –3. Even  of prey ,6. Multi of prey ,8, seem  of prey ,10. The  of prey , coupled of prey , certainanterior blind area occurs because there is a gap between the eyes that lie on either side of the mid-sagittal plane . It is this reliance on the beak that makes the avian case special in regards to the anterior blind area. Functionally, an anterior blind area creates a space around the beak where there is a lack of information that may be important for visually controlling the beak as it approaches a target. The longer the blind gap length, the longer the distance that birds would need to estimate  and the longer the time of uncertainty before the beak reaches the target .We propose that in bird species requiring visual control of the beak, the length of the blind gap would be relatively small to minimize the spatial and temporal uncertainty while interacting with the physical world. The benefit of reducing the blind gap length would be particularly pronounced in species that keep their eyes open and continuously accommodate their lenses to visually track their targets during pecking . OthOn an evolutionarily scale, the anterior blind area would be shorter when orbits are rotated forward in the skull, which would increase binocular field width ,24. AddiWe corroborated with empirical data the geometric relationship by which an increase in binocular field width is associated with a decrease in the anterior blind area length. As a result, we predicted that the binocular field would be brought closer to the beak, leading to a negative relationship between binocular field width and blind gap length. Because longer beaks extend further out from the head and are easier to see, having a long beak would also relax the pressure to bring the eyes forward to reduce anterior blind area length. Therefore, we predicted that species with longer beaks would also have longer anterior blind areas and thus narrower binocular fields.Given the large interspecific differences in foraging techniques and diets, we also made predictions for different relationships between the length of the anterior blind area and beak morphology depending on foraging requirements, sorting species into four groups . We predicted that pecking foragers would be able to tolerate some amount of blind area in front of the beak tip because their food/prey items do not move (or move very slowly). Instead, they can use the symmetrically expanding optic flow-field in the direction of travel to anticipate time to contact , and seeWe predicted that species requiring more visual control of the beak  would use eye movements more to temporally manipulate the size of their anterior blind area compared to species that do not require considerable visual control of the beak. Consequently, species with more visual control of the beak would be able to see their beak tips  more often when they converge their eyes forward rather than when their eyes are in the resting position.Diurnal raptors, or birds of prey, share many characteristics with non-raptorial predators in terms of the visual information needed to detect and pursue prey ,26; howeIn this study, we tested these predictions for the first time using multiple species of birds and controlling for the effects of phylogenetic relatedness. We used published data on visual field configuration, and gathered beak and skull measurements from museum specimens.We compiled data from the literature on the binocular field width from 40 species of diurnal birds  until torbitale superius that horizontally aligned with the optic foramen. We also measured beak length (the posterior end of the nares to the beak tip) and skull width (widest point posterior to the orbits). All represented skull measurements are the average of multiple individuals with equal numbers of males and females within each species  package DendroPy (version 4.0.0) [We used phylogenetic generalized least squares (PGLS) regressions to test for correlations while controlling for phylogenetic effects in R (version 3.3.0) using the pgls function. The phylogenies used in the analyses were obtained from backbone . We genen 4.0.0) ,35.2 = F, which is appropriate because the numerator degrees of freedom is 1. The variables anterior blind area length and blind gap length were standardized for differences in head size by dividing each variable by skull width. Certain variables were log base-10 transformed to meet the normality assumptions of the model. If variables included a value between 0 and 1, then the entire variable was log transformed as log10(variable + 1 –min(variable)). Because binocular width and standardized anterior blind area length are geometrically associated to one another, they were never included as co-explanatory variables in the same regressions to avoid violating model assumptions. Standardized anterior blind area length and standardized blind gap length could be included together without violating assumptions (R2 < 0.6).PGLS is designed to work best with continuous explanatory factors. Therefore, comparisons between different foraging groups were carried out using dummy variables of 0 and 1 rather than discrete ‘pecking’ and ‘raptor’, for example. The only statistics not using PGLS were tests of whether tactile foragers had significantly more variation in blind gaps than other groups. Those tests were done using the var.test function in R. In all PGLS results, t-values given by R were converted to F-statistics using tConsidering all bird species studied and controlling for beak length , we founWe predicted that pecking foragers would not be able to see their own beak tip because they would not necessarily need to see their beak to visual control it. We predicted that non-raptorial predators would be able to see their beak tip because they have more active prey that are difficult to target. Putting these two predictions together, we also expected non-raptorial predators to be able to see more of their own beaks than pecking foragers. We actually found that both pecking foragers and non-raptorial predators could see their beak tips on average because they both have negative blind gap lengths . However, the degree to which the beak was visible to these two groups varied. Non-raptorial predators were able to see the distal 74% (±7%) of their beaks, leaving only the most basal regions near the nares in the anterior blind area. This was significantly more than in pecking species, whose beak visibility was limited to the tip of the beak  and the blind area that occurs behind the head. As the binocular field widens and the length of the anterior blind area shortens in front of the head, the blind area behind the head becomes wider, reducing overall visual coverage . ReducedIndeed, we find that eye movements play a crucial role for pecking foragers and non-raptorial predators, which both rely on visual control of the beak to some extent. On average, neither group can see their beaks when the eyes are at rest, but both groups gain beak visibility when the eyes are swung forward. The total proportion of their daily time budget that requires fine beak control is relatively low. Therefore, both groups make use of large amplitude eye movements to visualize the beak while foraging but maintain a large cyclopean field of view while vigilant. For example, pigeons can only see their beak when their eyes are converged, and behavioral experiments have shown that pigeons swing both eyes forward to a converged position when pecking at seeds . A recenCorvus moneduloides) and American crows (Corvus brachyrhynchos), have particularly wide binocular fields and can see their beak tips [If fine manipulation or identification of captured food items is required, then visibility of not only the beak tip but also the beak edges where manipulation occurs may be important. Starlings  and meadeak tips ,21; henceak tips . When theak tips . WithoutNon-raptorial predators, such as insectivores and piscivores, have the need to visually guide mobile prey into their beaks, consequently the anterior blind area length of species with shorter beaks is shorter, hence increasing binocular overlap, than species with longer beaks. Even more interesting is that non-raptorial predators can see a larger proportion of their beaks than pecking foragers, which underscores the relevance of beak visibility in prey capture, identification, and manipulation tasks. Cormorants, for example, use visual control of the beak to expertly manipulate fish so that their fins are pointing backwards to make swallowing easier .As predicted, we found that diurnal raptors had a longer blind gap between the beak and the start of the visual field than non-raptorial predators . Like otScolopax spp.), for example, use touch receptors concentrated in the tip of the beak to detect and capture invertebrates within soft substrates [Anas clypeata) have more comprehensive cyclopean visual coverage around the head than their visually foraging congeners, Eurasian Wigeon (Anas penelope). The extra visual coverage pays off behaviorally, allowing the shovelers to spend less time vigilant and more time foraging [We found that tactile/filter foragers have the greatest variation blind gap length  and havebstrates ,47, and bstrates  found thforaging . There aforaging .The literature to date has tried to understand avian binocular vision by comparing the size of the binocular field across different species, which has led to some fruitful hypotheses ,10,38. HS1 TableUnits for binocular field widths are in degrees. Unless specifically noted, values were calculated using converged binocular field widths. Units for the distance from focal point to peak tip, internodal distance, and skull width are in mm. Standardized parameters were divided by skull width to control for body size. Negative values of standardized blind gap indicate the ability to see the beak tip and positive values indicate the inability to see the beak tip. If no resting binocular state was reported in the literature, we calculated it as the extrapolated converged binocular–(0.33 * (extrapolated converged binocular—diverged binocular)) which is the proportion for starlings in Martin 1986.(DOCX)Click here for additional data file.


HGT) as additional, relative constraints. These HGT constraints improve the precision of phototroph divergence date estimates and indicate that stem green non‐sulfur bacteria are likely the oldest phototrophic lineage. Concurrently, crown Cyanobacteria age estimates ranged from 2.2 Ga to 2.7 Ga, with stem Cyanobacteria diverging ~2.8 Ga. These estimates provide a several hundred Ma window for oxygenic photosynthesis to evolve prior to the Great Oxidation Event (GOE) ~2.3 Ga. In all models, crown green sulfur bacteria diversify after the loss of the banded iron formations from the sedimentary record (~1.8 Ga) and may indicate the expansion of the lineage into a new ecological niche following the GOE. Our date estimates also provide a timeline to investigate the temporal feasibility of different photosystem HGT events between phototrophic lineages. Using this approach, we infer that stem Cyanobacteria are unlikely to be the recipient of an HGT of photosystem I proteins from green sulfur bacteria but could still have been either the HGT donor or the recipient of photosystem II proteins with green non‐sulfur bacteria, prior to the GOE. Together, these results indicate that HGT‐constrained molecular clocks are useful tools for the evaluation of various geological and evolutionary hypotheses, using the evolutionary histories of both genes and organismal lineages.Phototrophic bacteria are among the most biogeochemically significant organisms on Earth and are physiologically related through the use of reaction centers to collect photons for energy metabolism. However, the major phototrophic lineages are not closely related to one another in bacterial phylogeny, and the origins of their respective photosynthetic machinery remain obscured by time and low sequence similarity. To better understand the co‐evolution of Cyanobacteria and other ancient anoxygenic phototrophic lineages with respect to geologic time, we designed and implemented a variety of molecular clocks that use horizontal gene transfer ( The absence of photosynthetic machinery in either of these outgroups is parsimonious with oxygenic photosynthesis evolving within the Cyanobacteria/Melainabacteria/Sericytochromatia (CMS) Group after the divergence of Melainabacteria, either via a fusion event via HGT of photosystems from other groups  generate conflicts between individual gene histories. These HGTs can be used as an informative character for the evolutionary history of organismal lineages  and PATRIC (https://www.patricbrc.org/) databases based on their ability to represent the phototrophic  and outgroup  taxa selected for this study. BLASTp was used to search each genome for homologous genes of interest   phylogeny, the best scoring ML tree was inconsistent with previous reports on cyanobacterial phylogeny Data  Sericyt was usedny Table  are prov2.2phylobayes v3.3   were excluded the computation of the chronogram. The 95% highest posterior density (HPD) intervals were calculated from the “datedist” file . Instead, Gloeobacter violaceus PCC 7421 is the outgroup to other cyanobacterial taxa  by the earliest evidence of habitability (4.4 Ga zircons)  from within GNS to stem GSB Data  that was33.1The results of Phototroph Models A and D (see Table When the SahH and BchH HGT constraints are applied (Phototroph Model D), GNS and Cyanobacteria are the oldest phototrophic lineages, and date estimate precision is improved. In particular, the 95% HPD intervals of stem GNS divergence date estimates are reduced by >50% of the non‐HGT‐constrained HPD interval. When only the SahH or BchH HGT constraint is applied (Models B and C), the uncertainty in the age estimate of GNS is restored Table . The sen3.2−5 times the present atmospheric O2 level. Although commonly referred to as the Great Oxidation Event (GOE), this transition was not a discrete event signifying at the onset of O2 production. Various forms of geological  and geochemical  features in the rock record have been reported as evidence for oxygen production prior to the GOE [For a comprehensive review, see:  to 3563 Ma . The generous root prior (3.8 Ga to 2.45 Ga) of Gloeobacter Outgroup Model 1 led to the least precise date estimates of Cyanobacteria taxa . Although the influence of root priors on molecular clock analyses has been previously reported . Under this sampling, the root of the phylogeny is interpreted as the LBCA, and the stem and crown age of Cyanobacteria can be inferred. When internal calibrations are omitted, the generous root prior (3.8 Ga to 2.45 Ga) results in very young Cyanobacteria date estimates . When internal calibrations are removed from our phototroph models, the crown age of Cyanobacteria is estimated to be younger than GOE (2172 Ma) but the stem age of Cyanobacteria (2892 Ma) allows sufficient time for the evolution of oxygenic photosynthesis within the Cyanobacteria stem lineage prior to the peak of the GOE 2.33 Ma  whose RCs are located along the plasma membrane, cyanobacterial RCs are located within thylakoid membranes  and crown Cyanobacteria arise ~2.2–2.3 Ga  rather than sulfide ‐oxidizing relatives that lived in Fe(II)‐rich waters prior to the GOE. Today, ferruginous ocean analogs are rare; however, a stratified lake in Indonesia  or alternative modes of phototrophy are the 3.4 Ga fossilized mat structures restricted to the photic zone of the Buck Reef Chert  ) from the posterior date estimates of Phototroph Model D  or thylakoid membrane containing Cyanobacteria  most likely appear shortly after the GOE (~2.3 Ga) while crown GSB  emerge just after the loss of the banded iron formations from the sedimentary record ~1.8 Ga. These events represent major changes in global chemistry and, potentially, indicate expansions of crown bacterial taxa into new environmental niches. Further constraining and testing these age ranges via additional fossil calibrations and HGT events will prove essential in establishing the direction of ancient photosystem HGTs and more tightly linking the genomic and geological records. Click here for additional data file. Click here for additional data file.


Introduction: Presbycusis or age-related hearing loss (ARHL) is a ubiquitous health problem. It is estimated that it will affect up to 1.5 billion people by 2025. In addition, tinnitus occurs in a large majority of cases with presbycusis. Glutamate metabotropic receptor 7 (GRM7) and N-acetyltransferase 2 (NAT2) are some of the genetic markers for presbycusis.Objectives: To explore patterns of hearing loss and the role of GRM7 and NAT2 as possible markers of presbycusis and tinnitus in a Portuguese population sample.Materials and Methods: Tonal and speech audiometry, tinnitus assessment, clinical interview, and DNA samples were obtained from patients aged from 55 to 75 with or without tinnitus. GRM7 analysis was performed by qPCR. Genotyping of single nucleotide polymorphisms (SNPs) in NAT2 was performed by PCR amplification followed by Sanger sequencing or by qPCR.Results: We screened samples from 78 individuals (33 men and 45 women). T allele at GRM7 gene was the most observed (60.3% T/T and 33.3% A/T). Individuals with a T/T genotype have a higher risk for ARHL and 33% lower risk for tinnitus, compared to individuals with A/A and A/T genotype, respectively. Being a slow acetylator (53%) was the most common NAT2 phenotype, more common in men (55.8%). Intermediate acetylator was the second most common phenotype (35.9%) also more frequent in men (82.6%). Noise exposed individuals and individuals with ‘high frequency’ hearing loss seem to have a higher risk for tinnitus. Our data suggests that allele AT of GRM7 can have a statistically significant influence toward the severity of tinnitus.Conclusion: For each increasing year of age the chance of HL increases by 9%. The risk for ARHL was not significantly associated with GRM7 neither NAT2. However, we cannot conclude from our data whether the presence of T allele at GRM7 increases the odds for ARHL or whether the A allele has a protective effect. Genotype A/T at GRM7 could potentially be considered a biomarker of tinnitus severity. This is the first study evaluating the effect of GRM7 and NAT2 gene in tinnitus. Presbycusis [age-related hearing loss (ARHL)] is a universal feature of mammalian aging in which the auditory function is compromised, hearing thresholds increase, and frequency resolution gets poorer. As a result, in noisy environments speech-understanding deteriorates and temporal processing deficits in gap detection measures increase . In humastria vascularis, afferent spiral ganglion neurons and the central auditory pathways  are the most commonly used devices for treating mild-severe presbycusis. Electric-acoustic stimulation and active middle ear implants may also be suitable solutions for treating presbycusis .N-methyl-D-aspartate (NMDA) and alfa-amine propionic acid (AMPA), the latter being the most relevant receptor in physiological neurotransmission at auditory pathways. NMDA receptors are not essential for the auditory transmission, but they have been shown to be expressed in the cochlea after induction of tinnitus. Moreover, it has been demonstrated that the application of NMDA antagonists directly into the cochlear fluid can block salicylate-induced tinnitus in animals  and metabolic diseases . It was  animals .GRM7 encodes a metabotropic glutamate receptor subtype 7 (mGluR7), a G protein-coupled receptor regulating auditory nerve excitability. When bound by L-glutamate, mGluR7changes the configuration of adenylyl cyclase, which has implications in the metabolism of AMPc, control of cellular cycle, and normal functioning of central nervous pathways. mGluR7 plays a general role in glutamate synaptic transmission  and their detoxification and has been postulated to play a major role in the overall aging process and to significantly contribute to the ARHL. Oxidative stress in the inner ear, secondary to impairments in defense mechanisms caused by certain polymorphisms related to a battery of antioxidant systems, could make individuals more susceptible to ARHL .S-transferases (GST) has been demonstrated.In the adult inner ear, presence of several detoxification and antioxidant enzymes including catalase, superoxide dismutase, glutathione peroxidase, and glutathione N-acetyltransferase (NAT) are enzymes responsible for the detoxification of exogenic substrates via N-acetylation or O-acetylation. In humans, the catalytic activity by NAT isoenzymes NAT1 and NAT2 may be regulate by these substrate concentration. Both isoenzymes are highly polymorphic and catalyze many aromatic amines and hydrazine substances important for the balance of the oxidative status. In addition, NATs are known to be involved in the detoxification of harmful xenobiotics .Our sample included 78 older individuals . Although all included participants have presbycusis we will consider a subgroup with normal hearing because the adopted classification uses conversational frequencies.Exclusion criteria were considered: inability to understand and sign the informed consent due to a significant cognitive impairment, an uncompensated medical disorder that requires urgent evaluation or if the individual has a serious psychiatric disorder. Also individuals over 55 years who presented possible factors that may overlap the variables under study were excluded , history of ototoxic drugs use, massive noise exposure, a history of previous malignancy with chemotherapy, history of autoimmune disorders and neurodegenerative and demyelinating diseases.This study had the approval of the Ethical Committees from Hospital Cuf Infante Santo , Nova Medical School (n°65/2014/CEFCM) and the National Department of Personal Data Protection (authorization number:1637/2016).Accordingly we obtained the Institutional Scientific Review Board approval of the process for taking informed consent and overall study design. The study was conducted in accordance with the Declaration of Helsinki.® FTA® card technology, was obtained from every subject.Written informed consent, clinical and familial history, audiological evaluation and a blood sample, using WhatmanA questionnaire concerning epidemiologic data  was completed by the researcher through participant interview.®, Assens, Denmark audiometer  and TDH39 headphones fitted with noise-excluding headset ME70 and bone conductor B-71. Audiometry was performed at frequencies from 0.25 to 16 kHz . The category of Hearing Loss (HL) was defined according to the average threshold across 500, 1000, 2000, and 4000 Hz in the better ear as mild (21–40 dB), moderate (41–70 dB), severe (71–95 dB) or profound (>95 dB), from an average of thresholds at 500, 1000, 2000, and 4000 Hz in the better ear, according to the European Working Group Genetics of Hearing Impairment (After Hearing thresholds were determined by pure tone audiometry (air and bone) according to ISO 8253 and 389. The exam was performed in a soundproof booth using an Interacousticsnt After .Speech audiometry evaluation was obtained with headphones (using mp3 player), or in open field, where the evaluator was hiding his lips to prevent lip-reading. The number of disyllables that patient repeats correctly was recorded. This intelligibility threshold for two-syllable words intends to measure hearing sensitivity threshold through the intensity level identification in which the patient can correctly identify 50% or more of a disyllables list. On the other hand, the speech discrimination evaluates the lowest intensity level at which a listener can understand speech.Psychoacoustic assessment consisted of loudness match, pitch match, minimum masking level (MML) or Feldmann masking curves, residual inhibition, and loudness discomfort levels (LDL). The severity of tinnitus was evaluated using the Tinnitus Handicap Inventory THI; . THI comAdditionally THI is a self-administered instrument, easy to quote, to interpret and has good psychometrics properties .GRM7 gene was assessed by qPCR for A/A, A/T, and T/T genotypes, at rs11928865 SNP. Concerning NAT2 gene, rs1041983, rs1801280, rs1799929, rs1799930, rs108 and s1799931 were assessed by qPCR or by bidirectional sequencing of the target region in order to identify all the SNPs.Total genomic DNA was extracted from a blood sample on FTA cards using a commercial NZY Tissue gDNA Isolation Kit , strictly according to the manufacturer’s instructions. Molecular analysis of p = 0.05.We conducted a descriptive analysis for variables such as gender and age. The audiograms were analyzed considering the best ear (estimated based on the lowest average of frequencies of 0.5–4 kHz). We also evaluated the “high frequency” pure-tone average (PTA) at 2, 4, and 8 kHz . Chi-squAll the results were analyzed through logistic regression model, where age and gender where considered as control for all other variables.n = 45, 57.7%), presenting an average age of 64.1 ± 5.35 years old. For men , the mean age was 65.3 ± 5.89 years old (Table 1).Participants in our study were 78 older adults aged 64.6 ± 5.58 years old (range = 55–75 years old). Most participants were female  and 8 kHz (p = 0.031) when comparing male and female . There were significant differences between age groups regarding the average and median values of hearing thresholds at frequencies of 4 kHz (p = 0.003), 8 kHz (p < 0.001), 10 kHz (p < 0.001) and 12 kHz (p < 0.001) when comparing the different age groups .The average hearing threshold values (by gender and age) are shown in p = 0.009), 8 kHz (p = 0.011), 10 kHz (p = 0.018) and 12 kHz (p = 0.002) . For males statistically significant differences were observed between age groups at 8 kHz (p = 0.009), 10 kHz (p = 0.003) and 12 kHz (p = 0.004) .When comparing hearing thresholds between the different age groups, we found significant differences in females at 4 kHz (p = 0.022) and 4 kHz frequency (p = 0.028) .According to age and gender grouping and comparing males to females we found significant differences for hearing thresholds for the age group 55–60 years old for 1 kHz frequency (Table 1) shows that in subgroup 1, 18 (23.1%) individuals who had normal hearing thresholds at speech frequencies (0.5–4 kHz) but not tinnitus; subgroup 2, 23 (29.5%) individuals who had normal hearing thresholds at speech frequencies and tinnitus; subgroup 3, 10 (12.8%) individuals who had hearing loss but not tinnitus; and subgroup 4, 27 (34.6%) individuals who had hearing loss and tinnitus . There are no statistical differences in age or gender between those four subgroups.Distribution of the individuals according to the hearing loss and tinnitus presence (p < 0.001 at the majority of frequencies).We found statistically relevant differences between the four described groups which corroborates the logical of having chosen this subdivision of our study population.  between subgroups, either for the right ear or for the left ear. The differences were found between subgroups 4 or 3 and the subgroups 1 and 2 for PTA (0%), SRT (50%) and (100%) .There were significant differences in speech audiograms . In respect to having or not tinnitus we found statistical differences between those groups (p = 0.003) (for more details see Appendix 1). We found statistically significant differences (p < 0.001) when comparing the four subgroups described in Table 1 (for more details see Appendix 2).Because our study population represents older adult individuals with sensory presbycusis we evaluated the “high frequency” pure-tone average (PTA) at 2, 4, and 8 kHz. We compared the groups of individuals with and without tinnitus and the four subgroups  had tinnitus.Figure 5). The most prevalent were measles, hypercholesterolemia, noise exposure, mumps, smoking and hypertension, in a descendent order of frequency.We have determined the distribution of studied comorbidities in our tinnitus population (Table 3). We found no relevant association between statins intake and hearing loss.In our sample, 49 participants (62.8%) reported to have high blood values of cholesterol. Of those, 27 individuals (55.1%) were taking medication (statins) (Appendix 3). There was a significant association between tinnitus and statins intake in those individuals reporting hypercholesterolemia  for most participants tinnitus was bothersome, only 10 subjects had a slight handicap.Subgroups 2 and 4 included participants with tinnitus. Concerning tinnitus laterality, 33 of them reported to have a unilateral tinnitus (12 on the right ear and 21 on the left ear) and 17 participants have a bilateral tinnitus. According to THI score . The odds of developing presbycusis was significantly higher for males than for females .GRM7 gene at rs11928865 SNP refers to A or T alleles and contribute for three possible genotypes: A/A, A/T, or T/T. GRM7 data are presented comparatively (Table 4) with data for Iberian Peninsula and Europe in order to compare our population with others.Results for Some genetic specificity has been reported for different populations regarding deafness genes, and, interestingly, genotypes representativeness for the individuals of our sample were in accordance with values described in the European population as well as in the Iberian population.Analyzing these results and considering the hearing thresholds, no significant differences were found in males or females when the three genotypes were compared, however, some differences in the pattern of the curves on the audiogram can be seen.Figure 6) the variable severe tinnitus (n = 12) joins the severe and catastrophic grades. We found relevant statistical association between the presence of GRM7 and severe tinnitus . The results are present in the Table 5.Considering the Tinnitus Handicap Inventory scores . However, the odds of HL is higher in individuals presenting A/T (29%) or T/T (2%) genotype, than in A/A genotype. The same results were observed when controlling for age and gender, however, in this case the odds of HL in A/T genotype individuals was nearly 39% higher than for A/A genotype individuals. The odds of HL in T/T genotype was 15% higher than in A/A genotype.When considering genetic data, The relation between tinnitus and GRM7 gene was evaluated considering two groups, one defined as “having an A allele” (AA + AT) other defined as “not having an A allele” (TT). Results were not significant , and 26 males (40%). Rapid (R) phenotype was least common , followed by Intermediate (I) phenotype  and Slow (S) phenotype  (please see Table 6).Genetic analysis of n = 6) of the individuals being the allele 4 present in 56.9% (n = 37) of the genotypes. The genotype 6A/6A previously associated with presbycusis was found in 6.2% (n = 4) of the individuals being the allele 6A present in 23.1% (n = 15) of the individuals. The most common genotype is 5B/5B accounting for 50% (n = 11) of all the homozygous genotypes  the sample (The genotype 4/4 (considered as wild type) was observed in 9.1% .Considering the Tinnitus Handicap Inventory scores, we found significant association between severity of tinnitus (grades severe and catastrophic from THI) and the presence of Tables 8) where age, gender and noise exposure were considered in the models with the purpose of controlling for confounding. The independent variable in the model was severe tinnitus (n = 12), (the sum of severe and catastrophic grades from THI) .All the results were analyzed through logistic regression model  were having medication (statins) to control cholesterol levels. These results are in accordance with previous publications . In indiNoise exposure and “high frequency” hearing loss seems to influence the occurrence of tinnitus, those were two of the most statistical relevant findings in our study population, which is in accordance with previous literature .Figure 2). Our results show a significant age-dependent increase of hearing loss in about 13% for both genders, although the risk of developing presbycusis is about three times higher for men. This finding is consistent with a previous reports . Corroborating our results, GRM7 SNP A > T was significantly different between the two groups  phenotype was the least common, followed by the Intermediate (I) and Slow (S) phenotypes.Concerning GRM7 and severe tinnitus. The chance for having a severe grade of tinnitus (severe or catastrophic grades in THI) is 14,2 higher for those carrying the allele A/T compared to T/T. Probably in larger scale studies could be demonstrated the role of allele A/A that is the less frequent in our sample.We found relevant statistical association between the presence of the allele A/T of NAT2 when compared to intermediate acetylator.The odds of developing severe tinnitus was relatively higher in the presence of slow acetylator phenotype of GRM7 can have a statistically significant influence toward the severity of tinnitus. As well slow acetylator phenotype of NAT2 seems to have a similar influence . Nevertheless, those results should be interpreted with caution and future studies in larger scale are necessary to confirm this correlation.Our data suggests that allele A/T of GRM7, NAT2 and tinnitus.However, present data shows that genotype A/T and T/T present, respectively, a 70 and 33.3% lower risk of developing tinnitus, when compared to A/A genotype. No other studies were found relating GRM7 and NAT2 gene and the presbycusis and tinnitus in a population of Portuguese older adults.To the best of our knowledge, this is the first study on the association between Tinnitus was present in the majority of the presbycusis individuals.Age and gender significantly influence the risk for presbycusis but not for tinnitus. Overall hearing thresholds rates increase exponentially with age (9% per year), and the increment rate and speed were gender-specific, but this increasing rate and velocity are different for women and men.High blood pressure, thyroid diseases and hypercholesterolemia seem to have an effect on the hearing thresholds but no significant associations were found.Our findings agree with previously observed correlations between tinnitus, noise exposure, and “high frequency” hearing loss.GRM7 or NAT2 were found in our sample. Our results precludes a definitive clarification about the role of GRM7 as a possible genetic biomarkers for ARHL, although since the genotypes A/T and T/T have higher odds for HL than A/A genotypes, thus A allele could be pointed as protective biomarker for HL Nevertheless, the current state of knowledge regarding GRM7 impact in presbycusis is insufficient to make conclusions, and so, further large-scale studies are necessary to clarify this relation.No significant associations between presbycusis, tinnitus, and Considering tinnitus severity (according to THI), our results bring-up very innovative conclusions.GRM7 and slow acetylator phenotype of NAT2  are prone to develop a more severe form of tinnitus, that requires specific therapeutic interventions and ideally personally tailored.Our data suggests the tracks that can lead to the pathway of a tinnitus severity biomarker. Potentially individuals carrying the allele A/T of The occurrence of presbycusis is thought to be determined by genetic factors but can also be influenced by environmental or comorbidities effects, with a huge impact on quality of life and general health . HoweverInformation on family history and clinical epidemiological data may help the design and development of future clinical management plans for an increasing presbycusis population.HH conceived and designed this study and had contributions to all its stages. MAp and MA performed the statistical analysis. HH, MF, and HC contributed equally to all other stages of the manuscript development, drafted and revised the manuscript. DR worked with HH on interpretation of results and created appendices. DR created all audiometric figures. JP, MA, AS, DH, and GF provided consultative advice and revised the final manuscript.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.


A fundamental concept in biology is that heritable material is passed from parents to offspring, a process called vertical gene transfer. An alternative mechanism of gene acquisition is through horizontal gene transfer (HGT), which involves movement of genetic materials between different species. Horizontal gene transfer has been found prevalent in prokaryotes but very rare in eukaryote. In this paper, we investigate horizontal gene transfer in the human genome.From the pair-wise alignments between human genome and 53 vertebrate genomes, 1,467 human genome regions (2.6 M bases) from all chromosomes were found to be more conserved with non-mammals than with most mammals. These human genome regions involve 642 known genes, which are enriched with ion binding. Compared to known horizontal gene transfer regions in the human genome, there were few overlapping regions, which indicated horizontal gene transfer is more common than we expected in the human genome.Horizontal gene transfer impacts hundreds of human genes and this study provided insight into potential mechanisms of HGT in the human genome. The acquisition of genes from an organism other than a direct ancestor, which is called horizontal gene transfer (HGT), is well known in bacteria and unicellular eukaryotes –6. AlthoA recent study investigated the possibility of HGT in 26 animal species  and a simplified analysis in a further 14 vertebrates. Genome-wide comparative and phylogenetic analyses show that HGT in animals typically gives rise to tens or hundreds of active ‘foreign’ genes, largely concerned with metabolism. This analyses suggest that while fruit flies and nematodes have continued to acquire foreign genes throughout their evolution, humans and other primates have gained relatively few since their common ancestor [In this study, we analyzed the pair-wise alignment data between the human reference genome hg19  and 53 oWe started with the pair-wise blastz alignments between the human reference genome hg19  and 53 oFrom the pair-wise alignment data, we identified the human genome regions that were conserved between 12 non-mammalian vertebrates and the human genome. These regions were then compared to the mammalian genomes to create HGT regions. A human genome region is called a non-mammal conserved region if this region is aligned to at least two non-mammal genomes with an identity no less than 40% and the aligned human genome region length is longer than 1000 bps. For these non-mammal conserved regions, if a region can be aligned to at most 8 (<20%) of the 41 mammals with an alignment identity ≥40% and length of the aligned region longer than 40% of the human region, this region is defined as a predicted HGT region  between vertebrates and the human genome and the length coverage  between mammals and the human genome to calculate the number of predicted or candidate HGT regions.To verify the conservation levels of the sequences we extracted, we randomly sampled 50 sequences from our results and verified the sequences based on the existence of homologous sequence in several groups of vertebrates using the University of California Santa Cruz (UCSC) Comparative Genomics track  , 19, 20.Homo sapiens gene data from ENSEMBL database and converted them into bed format file. For each sequences dataset, we intersected the Ensembl Genes with these sequences to discover genes that had any overlap with the predicted HGT regions.We checked whether our sets of predicted HGT sequences overlapped with known coding genes in ENSEMBL database . We downWe extracted the transcript IDs from the overlapped Ensembl genes and searched these IDs through the Ensembl Human Gene sets table, in which these genes were classified as protein coding, pseudogenes, etc. .All involved genes were generated from Ensemble Genes  and enriUsing the Broad chromHMM  ChromatiWe used the RepeatMasker track from the UCSC Genome Browser to generate distributions of the different types of repeats, including SINEs, LINEs, LTRs, DNA repeats and simple repeats . We diviWe calculated the GC content and drew GC content curve for the HGT regions with a similar approach as the above section we did for repeats.In order to predict regulatory functions of our sequence, regulatory related histone modification and histone variants on the sequences in H1-hESC cell lines was investigated. One transcription related histone marks: H3K4Me1; and four regulation-related histone marks and variations: H2A.Z, H3K4Me3, H3K9Ac, and H3K27Ac  were appWe took the phylogenetic tree directly from the Comparative Genomics track “Vertebrate Multiz Alignment & Conservation (100 Species)” , 26. ForIn order to check the confidence levels of the HGT regions found in the human genome, we checked the possible transfer paths from other species to human. 22 HGT fragments  were aligned with blastn to the Nucleotide collection database of National Center for Biotechnology Information (NCBI), which consists of all nucleotide sequences available. We searched the Nucleotide collection database to find the homologous sequences of our HGT fragments in some species, which might be the media species for the HGT progress, such as parasites, bacteria or fungi.In our pipeline see , we compt-test p-value < 0.001).We located these HGT regions on the human chromosomes. The number of HGT regions varies from chromosome to chromosome and most of HGT regions are located on both ends of the chromosomes Table , Fig. 2.Since the HGT regions we predicted here happened after the split of primates from mammals, this discovery leads us to conjecture that the HGT fragments might be more likely to insert on both ends of the chromosomes.We found 642 Ensembl genes overlapped with predicted HGT regions and the types of genes were identified according to the Ensemble gene annotation. The most prevalent type of genes was “protein coding”, which covers 39.1% of HGT regions, followed by “lincRNA” (6.6%) and “antisense” (2.9%). The non-gene region covers 46.1% of the HGT regions. Background data (the whole human genome) showed that “protein coding” genes cover 40.7% of human genome, followed by “lincRNA” (7.0%), and “antisense” (3.7%). The non-gene regions cover 45.7% of human genome Table . Therefop-value < 0.05), we discovered one term of gene functions are significantly enriched. This term is metal ion binding (120 genes)  database and categorized these into functional groups and analyzed their enrichment in certain functions. With the modified Fisher Exact test (s) Table . OverallComparing the Chromatin States of predicted HGT regions and human genome, we found that, of the HGT regions, 61% were heterochromatin, 23% were transcription, and 6.8% were repressed. However, in the human genome, 72% were heterochromatin, 20% were transcription, and 1.6% were repressed. Table , Fig. 4.Using the RepeatMasker track, we calculated the percentage of HGT regions that were known repeats, as well as the proportion of each type of repeats . The percentage of repeats (average: 24%) in HGT regions was lower than that of the background region (average: 41%). The most prevalent type of repeats is “simple repeat”, occupying about 10% of the whole HGT regions, followed by “Low complexity repeat” at 4.5%. Background data shows, in human genome, the most prevalent types of repeats are LINE and SINE repeats, with minimal amount of “simple repeats” Fig. .Fig. 5aCompared to the flanking regions, the predicted HGT regions contains higher percentages of “simple repeat” and “Low complexity repeat”. But for the other types of repeats, such as “LTR”, “SINE” and “LINE” repeats, the percentage in HGT regions is lower than the upstream and downstream regions Fig. .We calculated the average percentage of CG content of predicted HGT regions and their 300 bps upstream and downstream regions. The average GC percentage of HGT regions is higher than the upstream and downstream regions Fig. .We calculated the average percentage of regulatory related histone modification signals in predicted HGT regions and their 300 bp upstream and downstream regions. Results showed no significant feature of histone modification in HGT regions Fig. .Our pipeline can find the HGT regions which have greater conservation with non-mammalian vertebrates than with most mammals. It is also important to note that we allow less than eight mammals containing homologous sequences with the human genome. Indeed, for most HGT regions we have found, some mammals do have homologous sequences with hg19 and most of them are the primates. This phenomenon indicated horizontal gene transfer happened between the ancestor of primates and the non-mammalian vertebrates.In order to further understand the HGT mechanism, we extracted homologous sequence of the predicted horizontal gene transfer sequences in mammals and non-mammalian species, constructed sequence phylogenetic tree and compared it with species phylogenetic tree. Comparison of two phylogenetic trees showed that, in most cases, two phylogenetic trees were not consistent. Then we compared the tree constructed from predicted HGT regions to the tree constructed from species genomes using the program SPRIT that estimated the number of required subtree prune and regrafts (SPR) to transform one tree into another. It was apparent from the SPRIT output that for most HGT regions, at least three or four SPRs are required to explain the homologous sequences' phylogenetic tree topology. Each SPR may correspond to at least one HGT event, therefore we conclude that at least three or four interspecies HT events have occurred during the evolutionary history of vertebrates. Besides, when we verify the possibility of these HGT events, we found that there were at least one HGT event between the ancestor of all or most primates and the non-mammalian.Lepidosauria (Lizard), and one fish (Lamprey). When we have searched the Nucleotide collection database to find the homologous sequences of our HGT regions in other species, we found that several fishes  have the homologous sequences, which suggests that fish might be the origin of this HGT region. Comparing the phylogenetic trees built by HGT fragments and species genomes, we discovered at least three predicted HGT events were required to explain the inconsistency. We thought there might be some HGT events happening between fish and Primates, between fish and Lizard or between Primates and Chinese hamster  and the length coverage (0) between mammalian vertebrates and hg19, we have extracted 22 fragments with lengths more than 1000 bps. These sequences are most likely to be HGT regions. We discovered that one of these fragments, range = chr11:71072901–71074379, has homologues sequence in four primates , one mammal (Chinese hamster), one 0% and thEchinostoma and Schistosoma. Both Echinostoma and Schistosoma can infect human and other mammals. Some species of Schistosoma can also infect birds and crocodiles. Other trematodes, which though have no homologous sequence of the predicted HGT fragments, can also infect the fishes [We were not able to remove the interpolation of gene lost. However, more evidence can be found to support HGT for these regions we identified. When we searched the Nucleotide collection database to find the homologous sequences of the predicted HGT fragments, we were able to find them in some species, such as parasite, bacteria or fungi, which might be the media species for the HGT progress. Results showed that most of our predicted HGT fragments have homologues sequences in human, fish, bird, poultry and livestock. Among the 22 most reliable predicted HGT regions, 6 have homologues sequences in at least one trematode parasite of human Table , such ase fishes . Some ofIn our pipeline, we can also change the similarity threshold  between vertebrates and hg19 and the length coverage  between mammalian vertebrates and hg19 to calculate the number of sequences and the total bases we extracted for HGT regions. With different threshold values, we have discovered different number of sequences. We have discovered 22 HGT regions when we set the similarity threshold to 60% and length coverage to 0. These 22 sequences are more likely to be the result of HGT events. We verified the sequences based on the existence of homologous sequence in several groups of vertebrates using the University of California Santa Cruz (UCSC) Comparative Genomics , finding 19 sequences have an abnormal conservation. When we lowered the similarity threshold to 40% and raised the length coverage to 40%, we have discovered 1,467 predicted HGT regions. In order to verify the result of 1,467 sequences, we sampled 50 of them to check their conservation levels manually in mammals and non-mammalian species using the (UCSC) Comparative Genomics track . We found 44 of the 50 regions (88%) have an abnormal conservation. Therefore, there might be hundreds or even more than a thousand predicted HGT regions in the human genome.We also analyzed the HGT potential of five invariant regions among all human genomes. None of our HGT regions overlapped with these five sequences, whose corresponding track displays from the UCSC Genome Browser can be found in Table There were 145 horizontal transfer genes found in Primates in a previous study . We compAccording to our previous knowledge of biology, horizontal gene transfer in prokaryotes is a common phenomenon, and the underlying mechanism has been extensively studied. In recent years, the studies of horizontal gene transfer in Eukaryotes has gradually increased, though these findings still remain controversy. At present, these previous studies still have many shortcomings, such as lack of evidences to prove these sequences are indeed derived from the horizontal gene transfer, few studies of underlying mechanism, incapability of finding common features of these HGT regions, and so on.Wolbachia) or some endoparasites  [Schistosomiasis share more orthologues with the vertebrates than they do with the ecdysozoans [Schistosomiasis) of humans and other Eukaryotes, which provided the evidence to prove horizontal gene transfer process we predicted is true.In this study, we not only identified 1,467 HGT regions in the human genome, but also analyzed the phenomenon of horizontal gene transfer in Eukaryotes. We analyzed the conservation of HGT regions in different Eukaryotic genomes to determine whether these sequences are derived from the horizontal gene transfer or not. The analysis of chromosomal locations of these sequences and repeat contents lead us to find some possible features of these HGT regions. We discovered the genes that had any overlap with our extracted sequences and we found most significant gene functions impact by HGT, by using gene function enrichment analysis. Comparing the phylogenetic tree built by homologous sequences with the species phylogenetic tree, we can predict the process of horizontal gene transfer. Besides, we also discovered the homologous sequences in the common parasite of humans and other eukaryotes. It has been reported that horizontal gene transfer in Eukaryotes need some media species to spread the genomic fragments. And these media species might be intracellular parasitic bacterium (anosoma) . One stuysozoans . So the In general, the majority of the HGT regions discovered by our pipeline is credible. The pipeline we built to analyze horizontal gene transfer in Eukaryotes is also reasonable. According to our previous discussion, we believe that horizontal gene transfer is more common than we expected in the human genome. This study will provide a reference for future study of the horizontal gene transfer in Eukaryotes.Hundreds of human genes were found to be more conserved with non-mammals than with most mammals. Few of these genes overlapped with known horizontal transferred genes. This indicated that horizontal gene transfer is more common than we expected in the human genome. This study provided insight into potential mechanisms of HGT in the human genome.


Müller glia are capable of de-differentiating and proliferating to become Müller glia-derived progenitor cells (MGPCs) with the ability to regenerate retinal neurons. One of the cell-signaling pathways that drives the reprogramming of Müller glia into MGPCs in the zebrafish retina is the Jak/Stat-pathway. However, nothing is known about the influence of Jak/Stat-signaling during the formation of MGPCs in the retinas of warm-blooded vertebrates. Accordingly, we examined whether Jak/Stat-signaling influences the formation of MGPCs and differentiation of progeny in the avian retina. We found that Jak/Stat-signaling is activated in Müller glia in response to NMDA-induced retinal damage or by CNTF or FGF2 in the absence of retinal damage. Inhibition of gp130, Jak2, or Stat3 suppressed the formation of proliferating MGPCs in NMDA-damaged and FGF2-treated retinas. Additionally, CNTF combined with FGF2 enhanced the formation of proliferating MGPCs in the absence of retinal damage. In contrast to the zebrafish model, where activation of gp130/Jak/Stat is sufficient to drive neural regeneration from MGPCs, signaling through gp130 inhibits the neurogenic potential of MGPCs and promotes glial differentiation. We conclude that gp130/Jak/Stat-signaling plays an important role in the network of pathways that drives the formation of proliferating MGPCs; however, this pathway inhibits the neural differentiation of the progeny. Müller glia in the retina can be reprogrammed into Müller glia-derived progenitor cells (MGPCs) with the potential to regenerate retinal neurons. The ability of Müller glia to produce MGPCs and regenerate neurons varies significantly between vertebrates. The MGPCs in the teleost fish have the ability to regenerate all types of neurons and restore visual function after injury5The transition of Müller glia into MGPCs involves de-differentiation, acquisition of progenitor phenotype, proliferation, and neuronal differentiation of progeny. A complex network of cell-signaling pathways coordinates Müller glia-mediated retinal regeneration; these pathways are beginning to be uncovered in the zebrafish and avian model systems78910In this study, we investigate how cell signaling through glycoprotein 130 (gp130)/Janus kinase/signal transducers (Jak/Stat) impacts the formation, proliferation, and differentiation of MGPCs in the chick retina. In the zebrafish retina, knockdown of Stat3 inhibits MGPC-formation in damaged retinas11in vivo.Müller glia in the avian retina can undergo wide-spread de-differentiation and proliferation in response to retinal injury or growth factors, namely FGF22314Gallus gallus domesticus) were obtained from Meyer Hatchery . Chicks were housed in a stainless steel brooder at about 25 °C and received water and Purinatm chick starter ad libitum.The use of animals in these experiments was in accordance with the guidelines established by the National Institutes of Health and the Ohio State University. All experiments were approved by the Institutional Animal Care and Use Committee at the Ohio State University. Newly hatched wild type leghorn chickens . To ensure that primers were not amplifying genomic DNA, identical reactions were ran without including reverse transcriptase. Primer-BLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast/) was used to design primer sequences, and their predicted product sizes and sequence information is included in tmTaq (Invitrogen) and an Eppendorf thermal cycler. To verify predicted product sizes PCR products were run on an agarose gel. All experiments include sample sizes of n ≥ 4, per time point, and 3 replicas per sample. Significance of difference  was determined by using a Mann-Whitney U test.RNA was extracted from retinas using the Trizol protocol (Invitrogen). Genomic DNA was removed from samples by following the DNA Free kit protocol from Ambion. cDNA was synthesized from the extracted mRNA using oligodT primers and SuperscriptOcular tissues were fixed, sectioned, and labeled similar to previous descriptions20In Situ Cell Death Kit .To identify dying cells that had fragmented DNA, we followed the manafacturer’s instructions using the Clodronate-liposomes were made as previously described2223Cell counts were performed on representative images and consistently made from identical regions of the retina to avoid region-specific effects. Central retina was determined to be 20° of the posterior pole of the eye, with a radius of approximately 2 mm. Peripheral retina was designated as 3 mm annual radius from the peripheral retinal margin. The chick retina is approximately 13 mm across.ImagPro 6.02 (Media Cybernetics) was used to quantify immunofluorescence as previously described202627Since pStat3 was present exclusively in the nuclei of Müller glia see , we wereSignificance of difference between two treatment groups accounting for inter-individual variability  was determined by using a two-tailed, paired t-test. Significance of difference  was determined between two treatment groups by using a two-tailed, unpaired t-test or a Mann-Whitney U test. GraphPad Prism 6 was used for statistical analyses. All experiments contained sample sizes between 4 and 8 animals.AP, a marker specific to avian Müller gliaStat3 is the primary effector of Jak/Stat-signaling and phosphorylation of Stat3 results in translocation to the nucleus and transcriptional regulationcntfr) and gp130 following NMDA-treatment. cntfr was rapidly up-regulated at 4 hrs after NMDA-treatment, and was down-regulated at 24, 48, and 72 hrs after treatment  and the transcriptional effector (Stat3) influenced the formation of MGPCs. To test this, we utilized JSI-124, which inhibits the phosphorylation of JAK2notch1 and dll4, whereas levels of hes5, ascl1a, and gli2 were unaffected , Hedgehog-signaling , and the transcription factor ascl1a are known to be up-regulated and are necessary for the formation of MGPCs in damaged retinas Intraocular injections of CNTF are known to induce GFAP-expression in Müller glia in chick and rodent retinas3940AP-positive Müller glia  and robust induction of pStat3 in TOPler glia . In addiler glia . Similarler glia . This isler glia . Intraocr glia 20. The FGFr glia 20, suggestgp130, cntfr, and cntf .


Valuable insights into the role played by visual experience in shaping spatial representations can be gained by studying the effects of visual deprivation on the remaining sensory modalities. For instance, it has long been debated how spatial hearing evolves in the absence of visual input. While several anecdotal accounts tend to associate complete blindness with exceptional hearing abilities, experimental evidence supporting such claims is, however, matched by nearly equal amounts of evidence documenting spatial hearing deficits. The purpose of this review is to summarize the key findings which support either enhancements or deficits in spatial hearing observed following visual loss and to provide a conceptual framework that isolates the specific conditions under which they occur. Available evidence will be examined in terms of spatial dimensions  and in terms of frames of reference . Evidence suggests that while early blind individuals show superior spatial hearing in the horizontal plane, they also show significant deficits in the vertical plane. Potential explanations underlying these contrasting findings will be discussed. Early blind individuals also show spatial hearing impairments when performing tasks that require the use of an allocentric frame of reference. Results obtained with late-onset blind individuals suggest that early visual experience plays a key role in the development of both spatial hearing enhancements and deficits. Our sense of vision provides us with the most detailed information about the spatial configuration of our environment. This visual dominance stems in part from the brain receiving high-resolution spatial information directly from the retina that is coded topographically throughout the visual pathway. While other modalities extract spatial information in a similar manner , they are body-centric and do not provide reliable information beyond personal and peripersonal space . However, there are exceptions to this rule, such as when using sensory-substitution devices to translate visual information into tactile input that can be perceived, for instance, on either the tongue or the back e.g., . The audsensory compensation hypothesis whereby blind individuals develop exceptional perceptual abilities within their remaining sensory modalities to compensate for the visual loss . In contrast, however, the blind display more accurate localization in peripheral auditory space, particularly for sound sources straddling the interaural axis .A seminal study identified a marked difference in spatial hearing ability between early blind and sighted individuals when having to localize sounds under monaural listening conditions . The spectral cue hypothesis, however, is challenged by spatial hearing findings in the vertical plane. Although localization ability in the vertical plane is also believed to rest primarily on spectral cues , early bAuditory depth perception has not been as extensively studied as localization, but there are nonetheless some emerging trends  in sighted individuals can improve auditory localization  is at odThe author confirms being the sole contributor of this work and approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Partial transection (PT) of the optic nerve is an established experimental model of secondary degeneration in the central nervous system. After a dorsal transection, retinal ganglion cells (RGCs) with axons in ventral optic nerve are intact but vulnerable to secondary degeneration, whereas RGCs in dorsal retina with dorsal axons are affected by primary and secondary injuries. Using microarray, we quantified gene expression changes in dorsal and ventral retina at 1 and 7 days post PT, to characterize pathogenic pathways linked to primary and secondary degeneration.Cryba1, Cryba2 and Crygs, were significantly downregulated in injured dorsal retina at days 1 and 7. While Ecel1, Timp1, Mt2A and CD74, which are associated with reducing excitotoxicity, oxidative stress and inflammation, were significantly upregulated. Genes associated with oxygen binding pathways, immune responses, cytokine receptor activity and apoptosis were enriched in dorsal retina at day 1 after PT. Oxygen binding and apoptosis remained enriched at day 7, as were pathways involved in extracellular matrix modification. Fewer changes were observed in ventral retina at day 1 after PT, most associated with the regulation of protein homodimerization activity. By day 7, apoptosis, matrix organization and signal transduction pathways were enriched. Discriminant analysis was also performed for specific functional gene groups to compare expression intensities at each time point. Altered expression of selected genes  and proteins  were semi-quantitatively assessed by qRT-PCR and immunohistochemistry respectively.In comparison to uninjured retina There was an acute and complex primary injury response in dorsal retina indicative of a dynamic interaction between neuroprotective and neurodegenerative events; ventral retina vulnerable to secondary degeneration showed a delayed injury response. Both primary and secondary injury resulted in the upregulation of numerous genes linked to RGC death, but differences in the nature of these changes strongly suggest that death occurred via different molecular mechanisms. Traumatic injury to the central nervous system (CNS) is the direct damage of brain or spinal cord tissue by a physical insult. Trauma also disrupts the physiological activity of homeostatic systems and auto-regulation of blood flow to intact regions, and triggers diverse inflammatory responses that together create a toxic environment conducive to delayed, secondary degeneration of tissue not initially affected by the trauma. This secondary degeneration results in more widespread pathology and even greater functional loss. Evidence suggests that secondary degeneration is a common factor following trauma to the brain  and spinAn understanding of the degenerative sequelae in neurons and adjacent glia following an initial injury is enhanced by being able to distinguish between neural tissue containing directly axotomized neurons and tissue containing intact, but subsequently vulnerable, neurons. Partial transection (PT) of the dorsal aspect of the rat optic nerve (ON) in Piebald Viral Glaxo (PVG) rats is now an established CNS model to simultaneously study a primary injury response and secondary degeneration following trauma –8. This 2+ overloading and increased reactive species . Further assessments at a later time point, confined to the ganglion cell layer, would allow more direct comparison of RGC death associated genes in primary and secondary degeneration. Taken together with previous findings, we demonstrate, following CNS injury, responses to primary and secondary degenerative events are genetically and mechanistically different, although in the retina at least, RGC viability is eventually compromised throughout. The complex molecular events that trigger secondary degeneration after CNS injury highlight the limited and narrowing time window for therapeutic intervention.Understanding the propagating genetic changes and molecular pathways following axonal lesion is key to the characterization of pharmacological intervention. Optic nerve injury neatly represents n trauma . In concS1 TextA: GSEA output on enriched functional groups comparing retina from dorsal day 1 PT to uninjured dorsal retina.B: GSEA output on enriched functional groups comparing retina from dorsal day 7 PT to uninjured dorsal retina.C: GSEA output on enriched functional groups comparing retina from ventral day 1 PT to uninjured ventral retina.D: GSEA output on enriched functional groups comparing retina from ventral day 7 PT to uninjured ventral retina.(DOCX)Click here for additional data file.


Despite recent work characterizing stem cells within proliferating infantile hemangioma, the expression of STAT proteins, well documented for their roles in stem cell signaling, has not been investigated. 3,3-Diaminobenzidine and immunofluorescence immunohistochemical staining revealed expression of pSTAT1, pSTAT3 and pSTAT5 in proliferating infantile hemangioma samples with the strongest expression of pSTAT3. There was reduced expression of these pSTAT proteins in the involuted infantile hemangioma samples. Western blotting confirmed the identification of all these three proteins in proliferating infantile hemangioma. It is therefore not surprising that the phosphorylated/activated forms of these proteins are relatively abundantly expressed in proliferating, in comparison to involuted infantile hemangioma samples. We speculate that the reduced STAT activation, as infantile hemangioma involutes, is a reflection of the depletion of the abundant stem cells within proliferating infantile hemangioma, as the lesion involutes.We have recently demonstrated the expression of embryonic stem cell markers on the endothelium of infantile hemangioma, a functional hemogenic endothelium with the capacity for primitive erythropoiesis  Infantile hemangioma (IH), the most common tumor of infancy, affects up to 10% of infants with a predilection for female, Caucasian and premature infants . It typiin vitro (We have recently documented the expression of embryonic stem cell (ESC) markers OCT4, signal transducer and activator of transcription 3 (STAT3) and stage-specific embryonic antigen-4 (SSEA-4) on the endothelium of proliferating IH . The endin vitro , 10.The STAT family acts as intracellular messengers in a variety of roles in stem cells and more specifically the hematopoietic stem cell (HSC) populations, including precursor expansion . Latent In the context of hematopoiesis, STAT1 signaling via IFN-α stimulation controls dormant HSC’s entry into the cell cycle  whilst SDespite recent advances in the characterization of the stem cell populations within proliferating IH , 16 the Eight proliferating and eight involuted IH samples obtained from patients undergoing surgical excision of IH were used for this study, which was approved by the Central Regional Health and Disability Ethics Committee (ref. no. 13CEN130). Written consents were obtained from all participants.n = 8) and involuted (n = 8) IH underwent 3,3-diaminobenzidine (DAB) IHC and immunofluorescence (IF) IHC staining on a Leica ASP200S Autostainer , using the Bond Polymer Refine Ready-to-use Detection Kit (Leica) for DAB staining. Slides were prepared for auto-staining by dewaxing and heat induced epitope retrieval using Bond Epitope Retrievals (Leica).4µm-thick formalin-fixed paraffin-embedded sections of proliferating , pSTAT3 , pSTAT5 , GLUT-1  and CD34 . Primary antibodies for IF IHC staining were detected using Vectafluor Excel anti-rabbit 594  and Alexa Fluor anti-mouse 488 .DAB IHC-stained slides were counterstained with hematoxylin prior to cover slipping and mounted using Surgipath Micromount mounting media (Leica). IF IHC-stained slides were mounted using Vectashield hardset mounting medium with 4', 6-diamidino-2-phenylindole (Vector Laboratories). Human tonsillar tissue was used as a positive control for pSTAT1, pSTAT3 and pSTAT5 and human placental tissue was used for CD34.DAB IHC-stained slides were viewed and the images were captured using an Olympus BX53 light microscope fitted with an Olympus DP21 digital camera . IF IHC-stained slides were viewed and the images were captured using an Olympus FV1200 biological confocal laser-scanning microscope .One snap-frozen proliferating and one involuted IH tissue samples from the original cohort of patients used for DAB IHC staining were used for Western blotting (WB). These tissue samples were processed as previously described  and tranProliferating IH showed plump, proliferating endothelial cells, organized into lobules with tiny lumina . This ceTo determine the localization of the cells expressing the pSTAT1, pSTAT3 and pSTAT5 proteins we performed IF IHC co-staining of these markers with the endothelial cell marker CD34 , green iTo confirm the protein identification by IHC staining, WB analyses of tissue lysates of a proliferating sample and an involuted IH sample for the presence of pSTAT1, pSTAT3, and pSTAT5 were performed. pSTAT1 was detected as a single band at the expected 91 kDa  in proliThe demonstration of the expression of pSTAT1, pSTAT3 and pSTAT5, and their localization in IH, is novel. The observation of phosphorylated/activated forms of these proteins being abundantly expressed in proliferating and markedly reduced or not expressed in involuted IH samples, may be due to down-regulation of STAT activation, associated with depletion of the relatively abundant stem cells in proliferating IH, as the lesion involutes  have been reported to be expressed by proliferating IH , 24. WidpSTAT5 expression within proliferating IH is not surprising in light of the demonstration of the expression of EPOR on the HE of proliferating IH . This stThis report presents novel findings of the expression of pSTAT1, pSTAT3 and pSTAT5 within proliferating IH. We speculate that expression of pSTAT1, pSTAT3 and pSTAT5 in cells of the endothelium and the interstitium of proliferating IH, may represent their involvement in putative stem cell signaling by both these populations, although further work is needed to clarify this. We propose that increased expression of these phosphorylated/activated proteins during proliferation and their reduction or absence during involution may reflect their involvement in stem cell maintenance, although conclusive proof of this is beyond the scope of this work.There are increasing reports of the role of stem cells in the pathogenesis of IH and this supported by the confirmed expression of markers such as alkaline phosphatase and CD133 on the endothelial cells of proliferating IH . The demThe phosphorylated/activated forms of STAT1, STAT3 and STAT5 are present in the endothelium and cells in the interstitium of proliferating infantile hemangioma.The phosphorylated/activated forms of STAT1, STAT3 and STAT5 are reduced or absent in involuted infantile hemangioma.This finding suggests roles for STAT1, STAT3 and STAT5 in the biology of stem cells within infantile hemangioma.Aspects of this work was presented at the Medical Science Congress, Queenstown, New Zealand, August 25–27, 2014; the New Zealand Association of Plastic Surgeons’ Annual Scientific Meeting, Queenstown, New Zealand, September 5–7, 2014; and the Plastic Surgery Congress, Brisbane, Australia, May 7–10, 2015. This work was also part of LS’s Bachelor of Medical Science thesis “The Activity of the JAK-STAT Pathway in Infantile Hemangioma and the Hemogenic Potential of Infantile Hemangioma Explant Derived Cells”, University of Otago, New Zealand.This study was approved by the Central Regional Health and Disability Ethics Committee (ref. no. 13CEN130). Written consents were obtained from all participants.LS, TI and ST came up with the study hypothesis and designed the study. LS, ET, TI, HB and ST interpreted the IHC staining results. LS analyzed the Western blotting data. LS, ET, PD, TI and ST drafted the manuscript. All authors approved the manuscript.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.


Whereas BiFC allows localisation of interacting partners, split-luciferase assay determines interaction strengths in vivo. Results demonstrate (i) interaction of Cnx2 and Cnx3 within the mitochondria and (ii) assembly of a biosynthesis complex including the cytosolic enzymes Cnx5, Cnx6, Cnx7, and Cnx1, which enables a protected transfer of intermediates. The whole complex is associated with actin filaments via Cnx1 as anchor protein. After biosynthesis, Moco needs to be handed over to the specific apo-enzymes. A potential pathway was discovered. Molybdenum-containing enzymes of the sulphite oxidase family interact directly with Cnx1. In contrast, the xanthine oxidoreductase family acquires Moco indirectly via a Moco binding protein (MoBP2) and Moco sulphurase ABA3. In summary, the uncovered interaction matrix enables an efficient transfer for intermediate and product protection via micro-compartmentation.Survival of plants and nearly all organisms depends on the pterin based molybdenum cofactor (Moco) as well as its effective biosynthesis and insertion into apo-enzymes. To this end, both the central Moco biosynthesis enzymes are characterized and the conserved four-step reaction pathway for Moco biosynthesis is well-understood. However, protection mechanisms to prevent degradation during biosynthesis as well as transfer of the highly oxygen sensitive Moco and its intermediates are not fully enlightened. The formation of protein complexes involving transient protein-protein interactions is an efficient strategy for protected metabolic channelling of sensitive molecules. In this review, Moco biosynthesis and allocation network is presented and discussed. This network was intensively studied based on two  Molybdenum (Mo) belongs to the group of essential metals like iron, zinc, manganese, or copper, which are used as cofactors or in prosthetic groups. These metals ensure redox enzyme functions in all organisms . As shown in Figure The Moco biosynthesis four-step reaction pathway as well as the biosynthesis enzymes are highly conserved and can be found in nearly all prokaryotes and eukaryotes. These enzymes are designated in plants by an abbreviation for The five Mo-enzymes found in plants are classified in two families based on the coordination chemistry of Mo in their active centre  in the cells depends on transformation and expression/degradation levels, respectively. Furthermore, results will be influenced by both reaction conditions as well as reaction partners depending on age, growing stage or vitality of the plant cells. These prerequisites have to be equalised by many experimental replications and the usage of appropriate negative and abundance controls. However, only in vivo interaction studies are able to verify the preconditions of a potential substrate channelling with so many partners in one protein complex.A protected Moco transfer requires tight protein-protein interaction between involved proteins within the plant cell. in vivo interaction studies are based on fusion proteins consisting of the POI's and a split reporter protein  Actin binding could increase the stability of the complex. Cnx1 first makes contact to actin followed by recruitment of the other components of the complex. A free diffusion in the cytosol could disturb these interactions and consequently the reaction efficiency. (ii) The anchoring could be important for positioning the whole complex near specific transporters to get the required substrates directly after import. One of these transporters is the mitochondrial ATM3 exporter, which transfers cPMP out of mitochondria. (iii) Another group of transporters provides molybdate for insertion into MPT by Cnx1. An efficient supply of molybdate is important for an uninterrupted reaction cascade inside the Moco biosynthesis complex. The animal Cnx1 homologue gephyrin clusters neuronal receptors at the postsynaptic membrane via microtubule binding. In an analogous manner, Cnx1 could cluster molybdate transporters on actin filaments for direct substrate channelling via protein interaction.The interaction network between the Moco biosynthesis complex and the Mo-enzymes forms a potential allocation pathway reducing the risk of oxygen damage of Moco. Mo-enzymes using di-oxo Moco have a direct interaction with the biosynthesis complex while Mo-enzymes using mono-oxo Moco receive the prosthetic group via an indirect pathway. Furthermore, the bypass of Moco via a binding protein and ABA3 leads to the hypothesis that di-oxo Moco is processed prior to insertion into the members of the XOR-family.For the first time, the complete Moco pathway interaction network has been identified in plants Figure  which miDK and RH were primarily involved in drafting the manuscript and DK produced the figures. C-KB and RM critically read the manuscript and improved the text. RRM and RH conceived the study and coordinated the work. All of the authors read and approved the final manuscript.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.


There are three times as many outer hair cells (OHC) as inner hair cells (IHC), yet IHC transmit virtually all acoustic information to the brain as they synapse with 90–95% of type I auditory nerve fibers. Here we review a comprehensive series of experiments aimed at determining how loss of the IHC/type I system affects hearing by selectively destroying these cells in chinchillas using the ototoxic anti-cancer agent carboplatin. Eliminating IHC/type I neurons has no effect on distortion product otoacoustic emission or the cochlear microphonic potential generated by OHC; however, it greatly reduces the summating potential produced by IHC and the compound action potential (CAP) generated by type I neurons. Remarkably, responses from remaining auditory nerve fibers maintain sharp tuning and low thresholds despite innervating regions of the cochlea with ~80% IHC loss. Moreover, chinchillas with large IHC lesions have surprisingly normal thresholds in quiet until IHC losses exceeded 80%, suggesting that only a few IHC are needed to detect sounds in quiet. However, behavioral thresholds in broadband noise are elevated significantly and tone-in-narrow band noise masking patterns exhibit greater remote masking. These results suggest the auditory system is able to compensate for considerable loss of IHC/type I neurons in quiet but not in difficult listening conditions. How does the auditory brain deal with the drastic loss of cochlear input? Recordings from the inferior colliculus found a relatively small decline in sound-evoked activity despite a large decrease in CAP amplitude after IHC lesion. Paradoxically, sound-evoked responses are generally larger than normal in the auditory cortex, indicative of increased central gain. This gain enhancement in the auditory cortex is associated with decreased GABA-mediated inhibition. These results suggest that when the neural output of the cochlea is reduced, the central auditory system compensates by turning up its gain so that weak signals once again become comfortably loud. While this gain enhancement is able to restore normal hearing under quiet conditions, it may not adequately compensate for peripheral dysfunction in more complex sound environments. In addition, excessive gain increases may convert recruitment into the debilitating condition known as hyperacusis. The audiogram is often considered the gold standard for assessing sensorineural hearing loss (HL). Individuals with pure tone thresholds of ≤20 dB HL would be classified as having normal hearing. However, there is growing awareness that the pure tone audiogram fails to detect certain forms of cochlear pathology and auditory processing deficits. This has led to the concept of “hidden hearing loss,” i.e., the realization that significant auditory perceptual deficits can exist in listeners with normal hearing thresholds, a condition that can exist when there is considerable IHC and/or auditory nerve fiber degeneration  can be used to interrogate the functional status of different structures in the cochlea and identify “hidden” damage to inner hair cells (IHC), outer hair cells (OHC), the IHC/type I auditory nerve fiber synapse, and spiral ganglion neurons (SGN). Sensorineural hearing loss is a complex phenomenon that not only involves the cochlea, but also numerous structures in the central auditory system capable of partially compensating for these cochlear deficits. Therefore, a more complete understanding of sensorineural hearing loss not only requires assessment with ECochG, but also examination of the neurophysiological changes occurring in the central auditory pathway. In this review, we will discuss our results from a comprehensive series of electrophysiological, neuroanatomical, behavioral, and neuropharmacological experiments in a chinchilla animal model of carboplatin-induced ototoxicity in which there is selective damage to the IHC and type I auditory nerve fibers that exclusively innervate the IHC. These studies illustrate how ECochG can be used to identify damage to the IHC and type I neurons that goes undetected  by the pure tone audiogram. Electrophysiological recordings from the inferior colliculus (IC) and auditory cortex (ACx) reveal how weak neural signals from a damaged cochlea are amplified as they ascend through the central auditory pathway. Finally, we discuss a few simple psychophysical tests we have shown can identify hearing deficits associated with damage to IHC and type I neurons.Cisplatin and other platinum based anti-cancer drugs are generally more toxic to OHC than IHC, with hair cell lesions generally progressing from the base toward the apex as the dose and duration of treatment increases  , which depend on OHC somatic electromotility , an AC receptor potential that mirrors the waveform of the acoustic stimulus, is predominantly generated by the OHC with only a small contribution from IHC , reflected as a sound-evoked DC shift near stimulus onset, is thought to be generated predominantly by the IHC receptor potential with a much smaller contribution from OHC , consisting of two negative peaks (N1 and N2), is the most widely studied component of ECochG. The CAP is most effectively elicited by acoustic stimuli with rapid rise time and is thought to reflect the synchronized onset response of type I auditory nerve fibers Dallos, . Since tHigh impedance microelectrodes can be used to record the all or none spike discharges from single auditory nerve fibers as they leave the cochlea and enter the cochlear nucleus. Since each type I auditory nerve fiber contacts a single IHC, the neural output of a fiber reflects the activity from a discrete region of the basilar membrane. When tone bursts are used to measure the response of a single auditory nerve fiber, one can map out the frequency-intensity combinations that are just capable of evoking a response, the so-called frequency-threshold tuning curve , inferior colliculus (IC), and auditory cortex (ACx) of awake chinchillas before and after carboplatin treatment ; a plot of threshold vs. frequency in the presence of the masker defines the NBN masking profile  based on the neural responses seen in the damaged cochlea Figures . The larWS, DD, GC, EL, JW, KR, and BA finished the experiment. RS, WS, DD, GC, EL, and KR analyzed the results. RS, BA, and WS wrote and edited the paper.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.