Although the pupil light reflex has been widely used as a clinical diagnostic tool for autonomic nervous system dysfunction, there is no systematic review available to summarize the evidence that the pupil light reflex is a sensitive method to detect parasympathetic dysfunction. Meanwhile, the relationship between parasympathetic functioning and hearing impairment is relatively unknown.To 1) review the evidence for the pupil light reflex being a sensitive method to evaluate parasympathetic dysfunction, 2) review the evidence relating hearing impairment and parasympathetic activity and 3) seek evidence of possible connections between hearing impairment and the pupil light reflex.Literature searches were performed in five electronic databases. All selected articles were categorized into three sections: pupil light reflex and parasympathetic dysfunction, hearing impairment and parasympathetic activity, pupil light reflex and hearing impairment.Thirty-eight articles were included in this review. Among them, 36 articles addressed the pupil light reflex and parasympathetic dysfunction. We summarized the information in these data according to different types of parasympathetic-related diseases. Most of the studies showed a difference on at least one pupil light reflex parameter between patients and healthy controls. Two articles discussed the relationship between hearing impairment and parasympathetic activity. Both studies reported a reduced parasympathetic activity in the hearing impaired groups. The searches identified no results for pupil light reflex and hearing impairment.As the first systematic review of the evidence, our findings suggest that the pupil light reflex is a sensitive tool to assess the presence of parasympathetic dysfunction. Maximum constriction velocity and relative constriction amplitude appear to be the most sensitive parameters. There are only two studies investigating the relationship between parasympathetic activity and hearing impairment, hence further research is needed. The pupil light reflex could be a candidate measurement tool to achieve this goal. The pupil light reflex (PLR) is the reflex whereby a change in pupil size occurs in response to an increase of light intensity falling on the retina . Under tStudies using the pupil light reflex to evaluate PNS activity are well established. For example, it has been used to test patients with clinical signs of PNS dysfunction, especially related to cholinergic deficiencies like Parkinson’s Disease , AlzheimMany studies about the pupil light reflex are available nowadays, but to evaluate its sensitivity in the evaluation of PNS activity, especially for PNS dysfunction caused by PNS related conditions, a thorough review of more recent pupil light reflex studies is needed. Certainly Loewenfeld’s book  includesMore fundamentally, the autonomic nervous system plays a direct role in the human stress response. SNS is in control of the so-called ‘fight or flight’ response to stressors. Sympathetic activation, releasing noradrenaline from adrenal glands, is the typical reaction when the body faces a stressful situation . In contHearing impairment may have adverse effects on daily life functioning. Multiple studies suggest that hearing impairment is associated with psychosocial problems like depression or loneliness , 15, 16.Sympathetic innervation to the inner ear is well documented. SNS innervates the cochlea by regulating cochlear blood flow via the local adrenergic α-receptors. In addition, inner ear function is under the influence of sympathetic activity via the central noradrenergic pathway . In contThis paper presents a systematic review of the existing literature, performed in conformance with the PRISMA statement . We firsThe systematic review was performed in accordance with the guidelines provided by the PRISMA statement . The supThis study was divided into three sections, describing the evidence for the relationships between: PLR and PNS dysfunction, hearing impairment and PNS, and hearing impairment and PLR see . In the Section 1: Pupil Light Reflex and Parasympathetic Nervous System DysfunctionP(types of participants): Participants of any age with recognized parasympathetic-related conditions  were considered. As no definitive list of parasympathetic-related conditions was forthcoming, the list of conditions for inclusion was assembled retrospectively after the overall search was completed, as follows: for each condition which featured in the results of search #6 : Pupil (pupillary) light reflex/ Pupil (pupillary) light reaction/ Pupil (pupillary) light response/Pupil (pupillary) response only caused by light stimuli. Studies measured with ‘pupil cycle time’ were excluded, because this measurement is different from standard PLR. Drug studies using PLR as a testing tool for autonomic function were excluded, as these studies assume that PLR reflects PNS function and therefore do not test this hypothesis.C(types of control group): Adults with normal parasympathetic functions or proven differences in parasympathetic functioning.O(types of outcome measures): PLR parameters and their relationships (correlations) to the PNS activity.Section 2: Hearing Impairment and Parasympathetic Nervous SystemP(types of participants): People with hearing impairment. Hearing impairment was defined as loss of hearing ability. Studies on Deaf people without any residual hearing who use sign language and lip-reading only were excluded. People whose hearing ability was restored by hearing aid(s) or cochlear implant(s) were also considered as hearing impaired. No restriction was applied to the severity of hearing impairment.I(types of intervention): Physiological measurements of PNS activity like heart rate variability, skin conductance and blood pressure.C(types of control group): Adults with normal hearing.O(types of outcome measures): PNS activity, measured by physiological methods other than the PLR and their relationships (correlations) with hearing status.Section 3: Hearing Impairment and Pupil Light ReflexP(types of participants): see P(types of participants) in Section 2.I(types of intervention): Pupil (pupillary) light reflex/ Pupil (pupillary) light reaction / Pupil (pupillary) light response / Pupil (pupillary) response only caused by light stimuli.C(types of control group): Adults with normal hearing.O(types of outcome measures): PLR parameters and their relationships (correlations) with hearing status.In addition, the following selection criteria were also imposed on all three sections of the review process.No animal studies.The language of the study was restricted to English only.Studies were published in a peer-reviewed scientific journal.Only quantitative studies were included; qualitative studies and case reports were excluded.th October 2014 and there was no limitation with regard to the year of publication.Potential studies were identified by searching electronic databases and reviewing the reference lists of papers. Five electronic databases were selected to perform the search: PubMed, Embase, Cinahl, PsycINFO and Cochrane. The search result was last updated on 30Hearing Impairment, Pupil Light Reflex and Parasympathetic Nervous System as the three key search terms in accordance to the objective of this review . The following steps were performed in EndNote to refine the results: 1) we omitted duplicates from the retrieved publications; 2) we omitted the papers that were not written in English; 3) we omitted the results for which no abstract was available.The refined results were exported with the identification number, title, and abstract (no authors were listed) for each study. Three reviewers  independently screened the results during the first screening stage. Three response options  were used to decide whether to include a study in the next stage. Reviewers also categorized each study into the three sections. To ensure the reliability of selection, any disagreement between the reviewers was resolved by discussing the abstract and full text of the paper if required. After the first screening, the publications included were inspected closely again by the three reviewers based on the full text, and the eligibility criteria of each section. Publications that survived this second screening were moved to the data extraction stage.We performed data extraction for each of the three sections separately . To thisFinally, we formulated a hypothesis about the correlation between the two key search terms, as well as the direction  of the hypothesized correlation. If the result of the outcome measure was in line with the hypothesis, a “+” sign was added to the data extraction sheet. If the result showed correlation in the opposite direction, then a “-” sign was given. Absent correlations were coded with “0”.The extraction strategy of PLR and PNS dysfunction articles was as follows:Type of PNS dysfunctionNumber of participants for each groupGenderAgeInformation about the nature of the participants:PLR measurement evaluating the PNS dysfunction of different groups.Parasympathetic measurements other than PLRInformation about the intervention method:PLR parameters Sensitivity of PLR parameters, i.e. whether a certain parameter showed a significant difference between groupsIf a given condition is related to PNS dysfunction, then patients with this condition exhibit an attenuated PLR relative to healthy controls. In sub-groups of patients with diagnosed different levels of severity of the condition, more severe levels are associated with more attenuated PLR.Whether a study fulfilled the hypothesis: Correlation between PLR parameters and other PNS measurement methodsInformation about the outcome measure:AND PNS dysfunction, 9 belonged to hearing impairment AND PNS. We failed to find any eligible studies about PLR AND hearing impairment.In total, 2046 records were identified from the search of the five electronic databases. After removing duplicates, excluding records in languages other than English and discarding those records without any abstract, 1366 records remained for the first screening. We discarded 1219 records based on their abstract and title, leaving 147 records. Among those records, 138 belonged to PLR and PNS was included, for which only the electronic copy of the paper was available online earlier than our cutoff date. We included it as there were so few papers in that section. Finally, there were 36 papers for PLR and PNS dysfunction, two for hearing impairment and PNS and none for hearing impairment and PLR  maximum constriction velocity (MCV), (the maximum slope of the constriction); 2) maximum constriction acceleration (MCA), ; 3) absolute constriction amplitude (ACA), (the amplitude difference between baseline and minimum pupil diameter of the constriction); 4) relative constriction amplitude (RCA), (the ratio of ACA to baseline pupil diameter (BPD), expressed as a percentage); 5) latency, (the time between light stimulus onset and constriction onset); 6) constriction time, (the time from the end of latency period to the moment of maximum constriction). Specific values can be found in data extraction tables in the appendix. The data were first extracted by YW, then SK, AZ, TL, GN each randomly selected one study to extract the data and to compare their result with YW’s original result in order to check the data entries.Studies investigating each different health condition were grouped together. For each study and each available PLR parameter, we noted whether statistically significant differences were found between the patient group and healthy controls or between different patient groups. Note that due to differences in test designs and light stimuli, it is impossible to directly compare the absolute values of PLR parameters between studies.Parkinson’s Disease (PD) is a degenerative disease of the nervous system with motor dysfunction. It is associated with PNS dysfunction, mainly due to an ACh and dopamine imbalance caused by dopamine deficiency . Pupil aPatients with Alzheimer’s disease usually suffer from reduced synthesis of ACh, which is the main neurotransmitter of the PNS. We found four eligible studies which examined the validity of the PLR as a measure of cholinergic (parasympathetic) dysfunction , 34, 35.Multiple system atrophy is a degenerative neurological disorder with symptoms that are similar to, but usually more severe as those of PD . MicieliThe alteration of ANS function due to aging is well recognized , 38. ThiAutonomic neuropathy is a common disabling complication of Diabetes Mellitus (DM), and monitoring autonomic activity could help to identify the early onset of DM . PupillaPrevious studies suggest that obesity is usually associated with a decrease in cardiovascular PNS activity , 52. PihAutonomic abnormalities are frequently reported in patients with Schizoprenia , and pupMigraine is a common chronic debilitating neurological disease that causes severe headache. It usually involves some ANS symptoms like nausea, vomiting or diarrhea , and theA study by Drummond  suggesteAbnormality of ANS functioning is well recognized in arthritis disease . StudiesMeniere’s disease is a disorder of the inner ear that is known to cause hearing-related problems like tinnitus and hearing loss. Research by Yamada et al.  suggesteMultiple sclerosis is an inflammatory autoimmune disorder that can involve disseminated lesions of the central nervous system and lead to autonomic disturbances. Evidence from cardiovascular measurements suggests that PNS dysfunction is common among multiple sclerosis patients , 76. We Heart failure is usually associated with decreased PNS function, as reflected by HRV measures . One PLRFamilial dysautonomia is a rare genetic disease that affects development of the sensory and autonomic nervous systems, including part of the PNS neurons. Dutsch et al.  tested PWe identified two studies whose patient groups exhibited a variety of autonomic neuropathies  rather than one specific disease. Bremner and Smith  recorded2. Hearing Impariment and Parasympathetic Nervous SystemTen results were promoted after the first screening for this section. During the second screening, eight of them were discarded due either to a lack of a proper measurement of hearing status or a failure to evaluate relations between hearing impairment and parameters evaluating PNS activity.Hasson et al.  studied While Hasson et al.  mainly fand PNS. No studies were identified considering the association between hearing impairment and PLR.In total, 2046 articles were found from our primary systematic search, and 38 of them met the inclusion criteria and were included as relevant studies after the selection procedure. Among them, 36 addressed the use of PLR to measure PNS activity and the other two were about hearing impairment The first main purpose of this review was to assess the sensitivity of PLR as a method to evaluate PNS dysfunction, since we found that there is no up-to-date systematic review available to generalize the research in this field. Across the various types of PNS dysfunction in the survey, 30 out of 36 studies clearly stated their findings of PNS dysfunction or decreased PNS tone in the patient group on the basis of PLR measurements in their results or discussion sections. Another 2 studies , 63 failand PNS dysfunction survey. Although latency was used most often, MCV seems to possess a higher likelihood of detecting PNS dysfunction. ACA and RCA also appear promising in this regard, whereas MCA and Time to MCV are insufficiently studied to draw any conclusions.Different parameters of the PLR were adopted in different studies. Whereas the PLR taken as a whole is governed by both SNS and PNS, the constriction phase is mainly controlled by PNS, so the parameters relating to pupil constriction may be considered as PNS-related PLR parameters. Bearing in mind that all parameters were derived from the same raw pupillometry data recording, it is still worth knowing which PLR parameters are the best indicators of PNS dysfunction. The nature of PNS-related diseases is crucial as it is the determinant of PNS dysfunction. Therefore, we have categorized studies according to different types of diseases to evaluate the effectiveness of PLR parameters within the same disease or PNS dysfunction. Most diseases found at least one PLR parameter to show decreased PNS tone in the patient group. Nevertheless, there are 4 exceptions, and two of them were studies about obesity , 54. AltLatency (the time gap between light stimulus onset and start point of constriction) was found to be the most often-used PLR parameter, but not the most sensitive one to PNS dysfunction. It is worthwhile to pursue this point further. Most studies that adopted Latency claimed that this parameter was an uncontaminated indicator of PNS activity, and that PNS dysfunction usually resulted in a longer Latency. Many of these studies cited the paper of Pfeifer and co-workers  that desMCV, ACA and RCA, on the contrary, are under direct control of the PNS system, and a reduced light reflex could be explained by reduction of activity in preganglionic parasympathetic fibers and ciliary ganglion neurons . One mayAnother perspective on the utility of PLR for detecting PNS dysfunction is to examine the correlation of PLR parameters with other established PNS measurement methods. For example, HRV parameters were significantly correlated  with PLR parameters in healthy athletes during exercise . Four stAlthough the technical demands of PLR measurement are relatively straightforward, it is also worth mentioning that PLR is a test that might be influenced by many factors other than those one wishes to study. The characteristics of the light stimuli , gender Moreover, effort has been made to quantify the PNS and SNS input in PLR by constructing mathematic models. Usui and Hirata  and YamaThe second purpose of this systematic review was to investigate the relationship between hearing impairment and PNS. Only two relevant studies , 27 wereHowever, both papers failed to clarify any exclusion critieria for their participants regarding any autonomic dysfunction conditions, as we described previously. It should be stressed here that hearing impairment might have a link to some of these conditions. For example, evidence suggests that hearing impairment is about twice as common in adults with diabetes compared to healthy controls , and theOur search did not identify any studies connecting the PLR and hearing impairment. This is another major outcome of this study. Apparently, the method is still unknown in the field of Audiology, despite the fact that pupillometry equipment and techniques are already in use in audiological research to assess sympathetic activity or listening effort , 103, anThere is a risk of bias when only including studies mentioning PNS or similar terms in their title or abstract, because if a study failed to find any difference from PNS-related PLR parameters, there would be a lower probability for it being mentioned in the title or abstract.In order for a given disease to be included, it had to be ‘well-known’ that it is related to PNS dysfunction. To determine this relationship, we searched ‘PNS’ and the name of each condition via electronic database. Some studies were thus excluded because we could not find any direct evidence suggesting its association to PNS dysfunction. This procedure is not absolutely objective, and may have introduced bias to our results.Comparison of PLR parameters: In general, there are two basic elements needed to complete a PLR test: light stimuli and a device to record pupil size. The characteristics of the PLR are highly dependent on the type of the light stimulus. Intensity, duration, waveform and frequency of the light all contribute to the reflex pattern . HoweverWe did not perform any quality assessment on the included studies due to the rather broad topic covered in this review. This fact may generate risk and bias to the review as the quality of studies varies from one to another.This review only focused on the evaluation of chronic PNS dysfunction using PLR measurement. However, PLR is also used extensively in the evaluation of task-induced PNS activity due to short term stress exposure. For example, there are studies recording PLR during numerical counting  in orderOnly two papers discussing the relationship between hearing impairment and PNS were identified from our systematic search. This lack of evidence makes it hard for us to draw any conclusion on our second goal.The major outcomes of this systematic review are:This is the first systematic review of the evidence concerning the sensitivity of PLR as a method to evaluate parasympathetic dysfunction. The relationships between PLR and PNS dysfunction were evaluated by reviewing the sensitivity of PLR as a testing method to differences between patients with PNS-related dysfunctions and healthy controls. Most of the included studies found at least one PLR parameter to be significantly different between the patient group and control group.The PLR parameters most likely to be sensitive to PNS dysfunction appear to be the Maximum Constriction Velocity (MCV) and Relative Constriction Amplitude (RCA).We only found two studies investigating the PNS activity in people with hearing impairment. The evidence base is thus weak, but points to reduced PNS activity in people with hearing impairment.We found no studies in the existing literature connecting PLR and hearing impairment. We believe this systematic review can help to bridge the gap between PLR and hearing impairment and may show a new direction for future research.S1 Appendix(DOC)Click here for additional data file.S2 Appendix(DOC)Click here for additional data file.S3 Appendix(DOCX)Click here for additional data file.


CDKL5) gene has been associated with epileptic encephalopathies characterized by the early onset of intractable epilepsy, severe developmental delay, autistic features, and often the development of Rett syndrome-like features. Still, the role of CDKL5 in neuronal functions is not fully understood. By way of a yeast two hybrid screening we identified the interaction of CDKL5 with shootin1, a brain specific protein acting as a determinant of axon formation during neuronal polarization. We found evidence that CDKL5 is involved, at least in part, in regulating neuronal polarization through its interaction with shootin1. Indeed, the two proteins interact in vivo and both are localized in the distal tip of outgrowing axons. By using primary hippocampal neurons as model system we find that adequate CDKL5 levels are required for axon specification. In fact, a significant number of neurons overexpressing CDKL5 is characterized by supernumerary axons, while the silencing of CDKL5 disrupts neuronal polarization. Interestingly, shootin1 phosphorylation is reduced in neurons silenced for CDKL5 suggesting that the kinase affects, directly or indirectly, the post-translational modification of shootin1. Finally, we find that the capacity of CDKL5 to generate surplus axons is attenuated in neurons with reduced shootin1 levels, in agreement with the notion that two proteins act in a common pathway. Altogether, these results point to a role of CDKL5 in the early steps of neuronal differentiation that can be explained, at least in part, by its association with shootin1.In the last years, the X-linked cyclin-dependent kinase-like 5 ( CDKL5; OMIM 300203), located on the X-chromosome, has been associated with severe encephalopathy associated with X-linked infantile spasms and some Rett syndrome (RTT)-like features [During the last years, the cyclin-dependent kinase-like 5 gene . The C-terminus of human CDKL5  fused to the Gal4 DNA-binding domain and expressed from the inducible pB35 vector, was used as bait to screen a random-primed Human Adult Brain cDNA library constructed into pP6.The yeast two-hybrid screening was performed by Hybrigenics Services, S.A.S, Paris, France  using a fully automated procedure. A confidence score  was attributed to each interaction as previously described [113 million clones (11-fold the complexity of the library) were screened using a mating approach with Y187 ) and CG1945 -CyC1TATA-LacZ lys2::GAL1UAS-GAL1TATA-HIS3 cyhR) yeast strains. A total of 171 HisFlag-CDKL5 (107 kDa isoform) has been described elsewhere . pCAGGS-3/cm2 for immunostainings and 1x104/cm2 for western blots. For overexpression studies, hippocampal neurons were transfected at 0 days in vitro (DIV0) by nucleofection using the Amaxa Basic Neuron Small Cell Number Nucleofector Kit following the manufacturer’s instructions. Embryos at E17 were used in this case to make neurons more resistant to the nucleofection process. Infection of hippocampal and cortical neurons was performed by adding lentiviral particles to the neurons before plating or 10 h after nucleofection.Primary hippocampal cultures were prepared from embryonic day 16 (E16) CD1 mouse embryos, considering the day of the vaginal plug as E0, as described previously  and platImmunoprecipitation of endogenous CDKL5 from P7 mouse brains was performed by homogenizing the whole brain in lysis buffer , incubation on ice for 20 min and centrifugation at 100.000 g for 30 min at 4°C. The mouse brain extract (500 μg) was incubated overnight at 4°C with 20 μl purified anti-CDKL5  or unrelExogenously expressed Flag-CDKL5 was immunoprecipitated from transfected HeLa cells lysed in lysis buffer  and PhosStop (Roche)). The sonicated lysate was centrifugated and the obtained whole cell extract pre-cleared with Protein-G agarose (Thermo Scientific). The anti-Flag resin (Sigma Aldrich) was blocked with 5% BSA in PBS for 4 h where after it was added to the pre-cleared extract. After over night incubation the immunocomplexes were washed several times with wash buffer , once with PBS and separated on a 8% SDS-PAGE.http://rsbweb.nih.gov/ij/).Primary neurons were fixed in 4% paraformaldehyde for 10 min, blocked for 1 h , then incubated overnight at 4°C with the primary antibody in phosphate buffer, 5% horse serum and 0.2% Triton X-100 and finally with the corresponding secondary antibodies. Nuclei were stained with DAPI and the cells mounted for microscopy analysis at a Nikon Eclipse Ni microscope equipped with a Nikon Digital Sight DS-2MBWc camera. Neuronal polarization was analyzed by staining with Tau1 and MAP2 . Neurites longer than 100 μm in which the intensity of Tau-1 staining increased significantly along the proximal to distal axis were counted as axons. According to this parameter, neurons were classified either as lacking an axon (no axon) when none of the neurites could be considered an axon or with multiple axons when more than one neurite showed the properties of an axon. Image analysis was performed with Adobe Photoshop and IMAGE J (Cdkl5 and shootin1 (both full length) were used to stain for endogenous mRNAs. In situ hybridizations were run on slide mounted cryostat sections (12 μm) using a protocol based on a previous published paper [Antisense riboprobes against mouse ed paper .6 cortical neurons isolated from E16 mouse embryos were infected at DIV0 to silence CDKL5 expression. At DIV7, cells were washed and lysed in UTC buffer . Approximately 200 μg of extract were loaded on 7 cm IPG DryStrips with a linear 4–7 pH gradient and isoelectric focusing performed with an IPGphor II apparatus  according to the manufacturer’s instructions. Subsequent SDS-PAGE was performed using 8% gels and proteins were detected by western blotting. Phosphatase treatment was done by adding 100 units of Lambda Protein Phosphatase  to the extract diluted in 2 ml 0.1% SDS, 0.1 mM MnCl2, and 1x phosphatase-buffer. After an overnight incubation at 30°C the samples were concentrated with Ultracel 10K centrifugal filters (Millipore) and subjected to isoelectric focusing.2.5x10HEK293T cells, grown in 150 mm dishes, were cotransfected with the packaging vectors pVSV-G, pMDL, pREV and either pLL3.7-shCDKL5#1, pLL3.7-shCDKL5#2, pLL3.7-shCDKL5#3, pLL3.7-shShootin#1, pLL3.7-shShootin#2, or pLL3.7-shLacZ using calcium phosphate. The viral particles were harvested 36 h post-transfection and concentrated by ultracentrifugation with SW32 rotor (Beckman Instruments) at 20.000 rpm for 2 h. Viruses were resuspended in PBS and stored at -80°C.t test and ANOVA two-way followed by Tuckey’s post-hoc test. Statistical significance was established as p < 0.05.Values are expressed as means ± standard error (SEM). The significance of results was obtained by Student’s To elucidate the neuronal functions of CDKL5 we searched for interacting proteins through a yeast two-hybrid screening. The C-terminal region of human CDKL5 (amino acids 299–1030) regulates its activities and serves as interfase with MeCP2, DNMT1, and PSD95 ,21,11. WTo evaluate when a physical association between the two proteins might exist in vivo, we compared their temporal expression pattern during brain maturation. By immunoblotting lysates of mouse brains at different developmental stages we observed that shootin1 was strongly expressed from E18 until P7 when its levels started declining becoming undetectable in the adult brain . In accoCdkl5 and shootin1 in the developing cortex by in situ hybridization  and residing in the cortical plate (CP). At E18 high levels of both shootin1 and Cdkl5 mRNAs were detected in the cortex, further suggesting that shootin1 and CDKL5 may be expressed by the same neurons, supporting the notion of their in vivo interaction.We then compared the expression pattern of dization . In lineTo assess in more details whether both CDKL5 and shootin1 are physically present in neurons during the first stages of neuronal differentiation, we analyzed their expression in primary hippocampal neurons prepared from E16 mouse brains and collected at different time points . Both shWe next analyzed the localization of the two proteins in cultured hippocampal neurons at the initial stages after plating . In stagAltogether, these data indicate the existence of a concurrent spatial and temporal expression profile for CDKL5 and shootin1, supporting the notion of a physical interaction between the two proteins.Shootin1 has a well-established role in regulating axon formation. Its interaction with CDKL5 prompted us to study the role of the kinase for neuronal polarization using loss- and gain-of-function approaches in hippocampal neuronal cultures. We first analyzed the effect of reduced CDKL5 levels for neuronal polarization. CDKL5, shootin1 or, as control, LacZ were silenced by infecting primary hippocampal cultures, prepared from E16 mouse embryos, with lentiviral particles expressing specific shRNAs. The efficient silencing of CDKL5 and shootin1 was verified by immunoblotting lysates prepared 96 h after infection with three shRNAs against CDKL5 and two against shootin1 . At DIV4We then analyzed how the loss of CDKL5 or shootin1 influenced axon specification by immunostaining with the axonal and dendritic markers, Tau1 and MAP2, respectively. Correctly polarized neurons were recognized as bearing a single axon with a strong distal staining for Tau1 and several minor MAP2 positive neurites, whereas others contained multiple axons or no axon . As expeWe next analyzed the effect of increased CDKL5 levels on neuronal polarization. To investigate the relevance of the catalytic activity of CDKL5 we also expressed a kinase-dead derivative K42R), carrying a mutation in a critical residue of the ATP-binding site [2R, carryThe above data suggest that CDKL5 might, at least partially, regulate axon formation through its association with shootin1. Therefore we asked whether the two proteins work in a common molecular pathway. We hypothesized that CDKL5 might promote axon specification only in the presence of shootin1, thus working upstream of shootin1. We therefore overexpressed CDKL5 in neurons in which shootin1 was silenced and analyzed neuronal polarization at DIV5. Neurons were nucleofected at the day of plating with CDKL5- or GFP-expressing vectors and, after another ten hours, infected with lentiviral particles expressing shShootin1#1 or shLacZ as control. As expected, a significant number of neurons expressing shLacZ and exogenous CDKL5 extended more than one axon  as internal standard for alignment. Interestingly, a reduction in signal intensity of the two most acidic spots, corresponding to the two shootin1 phospho-isoforms, was easily detected when CDKL5 was silenced . Consistull mice  was alteAltogether, these experiments confirm a model according to which CDKL5 works upstream shootin1, directly or indirectly affecting its phosphorylation.CDKL5 gene have been identified in patients with the early onset seizure variant of Rett syndrome and other neurological disorders sharing common features such as the appearance of intractable seizures during the first months of life, infantile spasms, hypotonia, and severe intellectual disabilities [CDKL5, might, to some extent, originate from such deficits. A delay in the migration of cortical neuronal precursors devoid of CDKL5 has already been reported [Cdkl5-null brains display reduced arborization [Mutations in the X-linked bilities . The biobilities . Howeverreported ,10. Thisreported ,10 have rization ,25.CDKL5 have recently been identified in patients with neurological deficits that include autistic traits [Here we show that proper CDKL5 levels are crucial also for axonal specification and elongation. The silencing of CDKL5 results in an increased number of neurons bearing no axon while both silencing and overexpression of the kinase are associated with the formation of supernumerary axons and reduced axonal length. This is the first time that a role of CDKL5 in neuronal polarization is reported. Nevertheless, it is relevant to mention that a role in regulating neurite outgrowth, rather than axon specification, has previously been reported for CDKL5 . We belic traits .Cdkl5-null cortices, reinforcing the idea that the two proteins work in a common molecular pathway.Since the molecular mechanisms by which CDKL5 regulates neuronal functions are far from understood, we aimed at identifying novel CDKL5 interactors that might help addressing this point. In a yeast two-hybrid screening we identified shootin1 as a novel interactor of CDKL5. Shootin1 is a brain-specific protein acting as a determinant of axon formation during the process of neuronal polarization. In unpolarized rat hippocampal neurons, shootin1 randomly fluctuates in the growth cones of multiple neuritis, eventually accumulating in a single neurite before its outgrowth as an axon . ImportaAs a possible scenario, we hypothesize that the absence of CDKL5 reduces the traction force by altering the phosphorylation state of shootin1, thus causing a reduction of axon length. Whether CDKL5 phosphorylates shootin1 directly or through an intermediary kinase remains to be revealed as well as the specific residues of shootin1 whose modification depends on CDKL5. It is interesting to note that Rac1 appears to be common to the pathways of both CDKL5 and shootin1. As mentioned, shootin1 phosphorylation is induced by cdc42/Rac1-dependent Pak1 activation and CDKL5 has been reported to regulate neuronal morphology acting upstream Rac1 ,24. In tEven if the main body of our experiments is in line with a role of CDKL5 in neuronal polarization through its association with shootin1, the picture is complicated by the fact that silencing of CDKL5 generates a mixed phenotype including both neurons devoid of axons and neurons with numerous axons. CDKL5 is very likely to influence different downstream pathways through its different interaction partners and may thus target different factors involved in regulating axon specification and outgrowth. In this scenario, we believe that CDKL5 influences neuronal polarization in part through shootin1 and in part in a shootin1-independent manner. Disruption of the shootin1-dependent mechanism generates neurons devoid of axons and a concomitant reduction in axon elongation of properly polarized neurons. This defect seems to be a mere delay in axon outgrowth and elongation since the percentage of neurons with no axon returns to control levels at DIV7, as also reported for neurons with reduced shootin1 levels . ConversCdkl5-null mice revealed that several signaling pathways are deregulated in its absence [Cdkl5-null mice [A kinome profiling of brain extracts from  absence . Interes absence . Accordiull mice , whereasull mice . In the ull mice  and it iull mice . FinallyAltogether, our data indicate a so far uncharacterized role of CDKL5 in the early phases of neuronal differentiation that may concur to the clinical features associated with mutations in this gene. Finally, both the association of shootin1 with a molecular pathway belonging to CDKL5 and the defects in neuronal migration previously associated with its deficiency  might maS1 Fig(A) Immunofluorescence analysis of primary hippocampal neurons expressing GFP (green) at stages 2–3 with anti-CDKL5 (red). Quantitative profiles showing the fluorescence intensities of GFP (green) and CDKL5 (red) from the soma to the distal tip of the neurites/axons indicated with asterisks are shown to the right. CDKL5 is present in the distal tip of the axon without showing a specific accumulation. (B) Hippocampal neurons were infected at the day of plating with lentiviral particles expressing two different shRNAs against CDKL5 or, as control, against LacZ. At DIV4, neurons were stained for CDKL5 (red); GFP is in green. The signal intensity of CDKL5 was analyzed with ImageJ and the mean values plotted in the graph to the right. n = 30.(TIF)Click here for additional data file.S2 FigImmuofluorescence of DIV4 hippocampal neurons expressing shRNAs against LacZ or CDKL5. Polarized neurons extend one Tau1-positive and MAP2-negative axon. Neurons silenced for CDKL5 present supernumerary axons (middle column) or do not extend any axon (right column). Tau1, MAP2 and GFP are in blue, red and green, respectively.(TIF)Click here for additional data file.S3 Figt test).Graphs showing polarization of hippocampal neurons expressing shRNAs against CDKL5 or LacZ at DIV4 and DIV7. The graphs show the percentage of polarized neurons, neurons with multiple axons and with no axon at the two different time points as means ±SEM . ***p<0,001, **p<0,01, *p<0,05. (Student’s (TIF)Click here for additional data file.S4 FigCdkl5-null mice were lysed in Tris pH 7.4, NaCl 150 mM, CHAPs 0.5%, EDTA 0.2 mM, PMSF 1 mM, inhibitors of proteases and phosphatases and treated or not with λ-PPase. Nucleic acids and lipids, possibly interfering with 2-DE, were removed by centrifugation in ultrafiltration concentration devices  at 15000 g to a final volume of 100 μl. Samples were subsequently diluted in UTC  and 200 μg of proteins were subjected to isoelectric focusing. The subsequent immunoblotting was performed first against shootin1 and next against neurofilament  without stripping the membrane for the anti-shootin1 antibodies. The NFL signal could thus be used as internal standard for alignment. n = 2.Cortices of P5 wild-type and (TIF)Click here for additional data file.


Two-component systems (TCSs) are abundant prokaryotic signaling pathways, whose evolution is of particular importance because of their role in bacterial pathogenicity. Comparative genomics can provide important insights into the evolution of these genes, but inferences are dependent on the relatedness of the compared genomes. This study investigated the relationship between evolutionary distance and TCS evolution in myxobacterial genomes, of which there are several sequenced examples, of varying relatedness, and which encode large numbers of TCSs.Myxobacterial TCS gene sets were compared, orthologues defined, and changes in TCS properties such as gene organisation, domain architecture and size identified.  Genome relatedness/evolutionary distance was found to have a large effect on the apparent frequency of evolutionary events affecting TCS genes, but not on the relative dominance of different types of mutations. Large (≥1 gene) indels were the most common changes, often giving rise to gene organisation changes. Smaller indels were also common, sometimes changing domain architecture, and/or leading to pseudogene formation. Individuality of myxobacterial TCS gene sets seems primarily due to lineage specific gene loss. However, there is also evidence of extensive acquisition of genes by lateral transfer, with gene duplication also creating new TCS genes.This study provides catalogues of myxobacterial TCS gene sets and their orthology relationships, benchmarked against genome relatedness. It also provides insights into the relationship between evolutionary distance and the inference of TCS estudies of TCS evolution beyond the myxobacteriavolution, which may be important for studies of TCS evolutiThe online version of this articleon beyond the myxobacteria.The online version of this article (doi:10.1186/s12864-015-2018-y) contains supplementary material, which is available to authorized users. Two-component systems (TCSs) are the dominant phosphorylation based signalling pathways in prokaryotes. The average eubacterial genome encodes ~50 TCS proteins , 2, and Comparative genomics and experimental approaches have shed light on the general principles of TCS evolution , 6, 7. FMyxococcales) are well known for their large numbers of TCSs, many of which have been characterised experimentally ), except that the LytTR family of RRs is expanded substantially in Ms (12 compared to two in each of Mx and Mf). As more genomes are completed it will become easier to identify more distant orthologues via ‘gap-bridging’ genomes. This will in turn improve deduction of the directionality of evolutionary changes such as indels, and allow us to create a more finely resolved timeline of myxobacterial TCS gene evolution.During the course of this study draft genomes became available for strains of and DZF1 , 49, howSo0157-2 , 51. BotFocusing on the TCS genes of myxobacteria, this study investigated the relationship between apparent evolutionary events, and genomic distance or ‘relatedness’. As genomic distance increases it becomes more difficult to identify TCS orthologues, and the relative frequency of evolutionary events increases. However, the relative frequency of evolutionary changes remain unaffected by scale (or lineage), and it seems that in the myxobacteria, as for other organisms, TCS gene evolution is dominated by large indels (often causing changes in gene organization), with smaller indels being less frequent . TCS genes are ‘born’ by duplication and acquisition through HGT, but the individuality of TCS gene sets appears to be dominated by lineage-specific gene loss.More generally, our results suggest that researchers should be mindful that the numbers of genomes compared can have a substantial effect on the apparent frequency of observed events. This is particularly true when comparing organisms that share different levels of ‘genetic relatedness’, which should not be assumed to always be consistent with the taxonomic assignments of compared organisms.


Arabidopsis thalianais the first model plant, the genome of which has been sequenced. In general, intensive studies on this model plant over the past nearly 30 years have led to many new revolutionary understandings in every single aspect of plant biology. Here, we review the current understanding of anthocyanin biosynthesis in this model plant. Although the investigation of anthocyanin structures in this model plant was not performed until 2002, numerous studies over the past three decades have been conducted to understand the biosynthesis of anthocyanins. To date, it appears that all pathway genes of anthocyanins have been molecularly, genetically and biochemically characterized in this plant. These fundamental accomplishments have made Arabidopsis an ideal model to understand the regulatory mechanisms of anthocyanin pathway. Several studies have revealed that the biosynthesis of anthocyanins is controlled by WD40-bHLH-MYB (WBM) transcription factor complexes under lighting conditions. However, how different regulatory complexes coordinately and specifically regulate the pathway genes of anthocyanins remains unclear. In this review, we discuss current progresses and findings including structural diversity, regulatory properties and metabolic engineering of anthocyanins in Arabidopsis thaliana. Anthocyanins are a group of colorful and bioactive natural pigments with numerous important physiological and ecological functions in plants. In general, anthocyanins attract pollinators and seed dispersers, protect plants from high light irradiation and scavenge free radicals produced in cells under stress conditions -6. In ad Arabidopsis thaliana is the first model plant, the genome of which has been sequenced. Over the past nearly three decades, intensive studies on this model plant have greatly updated our understandings in plant biology including the biosynthesis and functions of anthocyanins and other metabolites. In this report, we review and discuss the structural diversity, biosynthesis and metabolic engineering of anthocyanins in this model plant. pap1-D (production of anthocyanin pigment 1-Dominant) and 35S:PAP1 transgenic plants as well as red pap1-D callus cultures x3Lx6Lx3R in the R3 repeat of MYB proteins interacting with bHLHs. Site mutation studies confirmed that this motif is responsible for the interaction with bHLH proteins [Protein-protein interactions among bHLHs, MYBs and TTG1 have been demonstrated by different experiments. Yeast two-hybrid and pull down assays have provided evidence that GL3, EGL3 and TT8 interact with TTG1, MYB family proteins PAP1/PAP2 and bHLH proteins themselves ,75,88. Iproteins . The C-terminal region of bHLH proteins is suggested to be required for regulating anthocyanin biosynthesis. Sequence analysis has identified that the N-terminal of bHLH proteins contains a region interacting with MYB and an acidic region while the C-terminal includes a bHLH domain likely involved in the formation of homodimer and heterodimer ,90. A repap1-D plants and cultured them in vitro. Although these red cells were characterized by heterogeneity of pigmentation, no specific morphological differentiations, such as the formation of pavement cells and trichomes, occurred in the culture conditions [PAP1 and TT8 in red cells. In addition, qRT-PCR analysis showed the up-regulation of the expression of GL3 in red cells. Our experiments suggest that TTG1, GL3/TT8 and PAP1 likely form the only WBM complex that activates the high production of anthocyanins in engineered pap1-D cells. The component properties of different WBM complexes remain to be further elucidated. Although biochemical and genetic studies have shown that TTG1 (WD40), GL3/ EGL3/TT8 (bHLH) and PAP1/PAP2/MYB113/MYB114 (MYB) are components of potential WBM complexes ,60. The nditions . Genome-In Arabidopsis, in addition to regulating anthocyanin biosynthesis, WBM complexes are involved in the control of other physiological and developmental processes including trichome initiation, non-root hair cell fate determination and mucilage production in the seed coat. This is in contrast to the function of WBM complexes in maize which seems to only control anthocyanin biosynthesis ,56,71,74in planta. As described above, TTG1, bHLHs (GL3/ EGL3/TT8) and MYBs (PAP1/PAP2/MYB113/MYB114) can form different WBM complexes to activate anthocyanin biosynthesis. However, other proteins, such as CPC and MYBL2 which are 1R-MYB members, have been demonstrated to negatively control the activities of WBM complexes resulting in the decrease in the biosynthesis of anthocyanins shown in Fig. (3). Results from transient expression and protein interaction studies have suggested that CPC and MYBL2 compete with positive regulators PAP1/PAP2 to bind bHLH proteins and interfere with the formation of active WBM complexes thus negatively regulating the expression of pathway genes [MYBL2 has been shown to repress trichome development [3).The activities of WBM complexes can be affected by factors ay genes ,88,89. Iay genes ,94. The elopment . The C-telopment . A recenelopment . We propTAS4-siR81(-), which is derived from TAS4 and miR828, is a trans-acting siRNA. TAS4-siR81(−) and miR828 were shown to target PAP1/PAP2/MYB113 [PAP1 is increased in tissues. PAP1 has been demonstrated to activate the expression of TAS4 and miR828, which may further function in a feedback manner to target PAP1 and its homologs to reduce their expression [PAP1 expression through TAS4-siR81(−) and miR828. In addition, transgene silencing of PAP2 has been observed in homozygous transgenic tobacco plants and was suggested to be caused by small regulatory RNAs similar to TAS4-siR81(−) and miR828 in Arabidopsis [miR156. The SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factor targeted by miR156 has been demonstrated to negatively regulate the acropetal accumulation of anthocyanins in the inflorescent stem [DFR. SPL9 was suggested to negatively control the expression of anthocyanin pathway genes by competing with bHLH proteins for binding with PAP1. The high expression of miR156 indirectly positively regulates the expression of anthocyanin pathway genes. Small regulatory RNAs were recently uncovered to control anthocyanin biosynthesis through a mechanism of regulating the expression of the members of the WBM complexes. 2/MYB113 ,98. In ppression . These rbidopsis . Anotherent stem . SPL9 waAnthocyanin biosynthesis can be induced by various abiotic factors such as high light, low temperature, sucrose, nutrient depletion and phytohormones ,101-105.PAP1, PAP2 and bHLH genes GL3, EGL3 and TT8 were all induced by various light spectra [PAP1 overexpression alone was not sufficient for the activation of anthocyanin biosynthesis in the dark or under low light conditions [Light is one of the most important environmental factors affecting biosynthesis of anthocyanins. Strong light conditions can increase the production of anthocyanins ,16,83. Inditions ,16,83, wPAP1 [CHS, CHI, F3H and FLS genes in studies conducted under UV-containing white light. The LRUs have been characterized to include a MYB-recognition element (MRE) and an ACGT-containing element (ACE), the latter of which is recognized by bZIP proteins such as HY5 [In addition, light signaling components have been demonstrated to play important roles in controlling anthocyanin biosynthesis. HY5, a bZIP protein, is a positive regulator of photomorphogenesis and can be degraded by COP1 in dark-grown seedlings . In far-PAP1 . Furtherh as HY5 . PAP1. A QTL analysis has shown that the expression of PAP1 is responsible for sucrose-induced anthocyanin accumulation [PAP1 but not PAP2 [Sucrose has been demonstrated to regulate anthocyanin biosynthesis in plants and cell cultures. In general, treating Arabidopsis seedlings with increased levels of sucrose can enhance the production of anthocyanins . A time mulation . In addinot PAP2 .SUC1 (SUCROSE TRANSPOTER1) showed less anthocyanin accumulation in response to sucrose [SUC2, a homolog of SUC1, has been shown to be involved in anthocyanin production in conditions of phosphate deficiency. The expression of SUC2 is highly up-regulated in the hypersensitive to phosphate starvation1 (hps1) mutant, which has an enhanced sensitivity to phosphate starvation [hps1 mutants have enhanced production of anthocyanins. Sucrose transporters appear to play a role in sucrose-induced anthocyanin biosynthesis. The mutants of  sucrose . In addiarvation . ConsequGL3, TT8 and PAP1 [SUC1 in roots [In addition, a crosstalk between sucrose and plant growth regulators has been shown to regulate anthocyanin biosynthesis. Jasmonate and cytokinin are known to induce anthocyanin production in plants; however, in the absence of sucrose, the regulatory functions of these plant hormones are not obvious ,114. Ethand PAP1 ,116. In in roots . PAP1 and PAP2 [PAP2 was shown to have a stronger response to nitrogen limitation than PAP1. This observation was supported by another experiment, in which the expression of PAP2 was strongly induced in senescing leaves treated by high sugar/nitrogen ratios [bHLH genes, GL3 but not EGL3 was highly up-regulated in rosette leaves of wild-type plants under nitrogen depletion [gl3 mutants accumulate much lower amounts of anthocyanins in rosette leaves under nitrogen depletion conditions compared with WT and egl3 mutants. A recent study suggested that the FRUITFULL (FUL) gene is also likely involved in the regulation of anthocyanin biosynthesis in response to nitrogen. The FUL gene regulates cell differentiation during fruit and leaf development in Arabidopsis [VmTDR4 has been identified to be an important regulatory gene in regulating anthocyanin accumulation during the ripening of bilberry fruits [PAP2 under nitrogen depletion conditions [PAP1 and PAP2 [Nitrogen sources can strongly control the biosynthesis of anthocyanins in Arabidopsis. A general trend is that seedlings produce low levels of anthocyanins in high concentrations of total nitrogen, in contrast, high levels of anthocyanins in low concentrations of nitrogen. Under nitrogen deficient conditions, seedlings have been reported to accumulate high levels of both anthocyanins and flavonols ,85. Pathand PAP2 ,117. In n ratios . For threpletion . In contbidopsis . Its homy fruits . Gene exnditions . Moreovenditions . The oveand PAP2 . PAP1, PAP2 and GL3 in response to JA [COI1 complex, which mediates the degradation of JA ZIM-domain (JAZ) proteins [COI1 complex, thus allowing the formation of the functional WBM complexes and leading to the production of anthocyanins. Jasmonate (JA) is an elicitor and signal molecule that mediates plant responses to pathogen infection, UV radiation and other abiotic stresses . JA can se to JA . It has proteins . JAZ proproteins . The potproteins . These r


Capacity assessments are unique because they examine the contribution of reaction-time (RT) as well as accuracy to determine the extent to which a listener efficiently combines auditory and visual speech cues relative to independent race model predictions. Multisensory speech integration ability was examined in two experiments: an open-set sentence recognition and a closed set speeded-word recognition study that measured capacity. Most germane to our approach, capacity illustrated speed-accuracy tradeoffs that may be predicted by audiometric configuration. Results revealed that some listeners benefit from increased accuracy, but fail to benefit in terms of speed on audiovisual relative to unisensory trials. Conversely, other listeners may not benefit in the accuracy domain but instead show an audiovisual processing time benefit.Research in audiovisual speech perception has demonstrated that sensory factors such as auditory and visual acuity are associated with a listener's ability to extract and combine auditory and visual speech cues. This case study report examined audiovisual integration using a newly developed measure of  McGurk effect in which incongruent or mismatched cues from the visual modality  influence auditory perception. Similarly, being able to see a talker's face under degraded listening conditions has been shown to facilitate both accuracy   2003) amFor adults with hearing loss, being able to see a talker's face can therefore prove exceedingly helpful in terms of enhancing accuracy. Facial movements—especially those associated with high-frequency sounds such as place of articulation—can “fill in” for auditory speech cues that have become degraded Erber, . As an eRelationships between low-frequency hearing acuity and speech recognition have also been reported. Erber , 2003)  2003) reC_I(t), that compares both accuracy and reaction times (RTs) to parallel independent race model predictions derived from auditory and visual-only speech recognition trials. We therefore argue that a complete procedure for assessing integration ability should include multiple relevant dependent measures, namely accuracy and RTs. Comparisons between obtained data and predictions derived from statistical models that serve as a null-hypothesis will also be included ,  and C_I(t) scores were associated with l pure tone thresholds as well as traditional accuracy-based assessments of audiovisual gain  was correlated with low-frequency thresholds.Listeners with auditory sensory deficits should adopt certain predictable strategies when processing audiovisual speech stimuli to maximize multisensory benefit. In terms of speed-accuracy strategies, some individuals may be slower on audiovisual trials relative to independent race model predictions in order to take advantage of visual speech cues. However, they may also be substantially more accurate and potentially show evidence for super-capacity and efficient integration . Additionally, we predict that these listeners should show greater audiovisual gain in open-set sentence recognition tasks . This scenario should often emerge in listeners with mild low-frequency and moderate high-frequency hearing difficulty. This type of hearing loss leads to mild degradation of certain vowel cues, and high-frequency information related to place of articulation. In these cases, auditory accuracy should be bolstered by visual speech cues. In fact, Altieri and Hudock  showed tC(t)) for listeners with mild to moderate high-frequency hearing loss, but normal low-frequency thresholds. This is because auditory-only processing in these listeners should be slower but not significantly less accurate compared to normal-hearing listeners (due to the availability of more low-frequency vowel cues). This will, however, allow for complementary visual cues to facilitate speech recognition in the processing time domain especially. Generally, there should be a range in which auditory-only recognition becomes difficult, but enough cues are present in the signal to permit the combination of redundant and complementary visual cues to facilitate accuracy, perhaps in addition to speed. In future research, we predict that subjects with hearing loss will generally show superior integration skills compared to normal-hearing listeners, either in the speed, accuracy domain, or both.Second, we predict a larger RT-capacity gain . The measured hearing loss could either occur for low-frequencies, high-frequencies, or both. Each of the five participants was a native speaker of an American English dialect. Each participant reported normal or corrected 20/20 visionAmbco 1000 Audiometer. Hearing thresholds were obtained in a sound attenuated chamber. Thresholds were obtained for 250, 500, 1000 (low frequencies), and, 2000, 4000, and 8000 Hz . Consistent with Bent et al.  software.Accuracy data from the 75 audiovisual (25), auditory-only (25), and visual-only (25) sentences listen in Supplementary Material were obtained from each participant. Trials were presented in separate blocks consisting of 25 audiovisual, 25 auditory, and 25 visual-only trials. The order of audiovisual, auditory, and visual-only block presentation was randomized across participants in an effort to avoid order effects. The stimuli in both experiments were presented to the participants using E-Prime 2.0 . Each movie was digitized and rendered into a 720 × 480 pixel clip at a rate of 30 frames per second. Similar to Experiment 1, the auditory component signal was degraded using the 8-channel sinewave cochlear implant simulator. The duration of the auditory, visual, and audiovisual files ranged from 800 to 1000 ms. A previous report demonstrated that the variation in the duration of the movies did not influence RTs; rather, linguistic factors such as the confusability of the auditory and visual phonetic cues proved to be a major factor affecting processing speed , 128 auditory-only trials, and 128 visual-only trials, for a total of 384 experimental trials. This portion of the experiment required 20–30 min to complete. Experimental trials began with a white dot on a gray background appearing in the center of the monitor. Each trial consisted of auditory-only, visual-only, or audiovisual stimuli. Auditory stimuli were played at a comfortable listening volume (approximately 70 dB SPL) over Responses were collected via button press using a keyboard. Each of the buttons, 1–8, was arranged linearly on the keyboard and was labeled with a word from the stimulus set. Participants were instructed to press the button corresponding to the word that they judged the talker to have said as quickly and accurately as possible. Responses were timed from the onset of the stimulus on each trial. Inter-trial intervals randomly varied on a uniform distribution between 750 and 1000 ms. On auditory-only trials, participants were required to base their response solely on auditory information, and on visual-only trials participants were required to lip-read to make the speeded response. Auditory-only trials were played with a blank computer screen. Similarly, visual-only trials were played without any sound coming from the speakers. Each listener received 48 practice trials at the onset of each experimental block to assist with learning the response mappings on the keyboard.AV) = p(A) + p(V) − p(A) * p(V) and obtained audiovisual (AV) scores. The actual AV Gain scores are also displayed (AVGain = p(AV) − max{p(A), p(V)}; cf. Altieri and Wenger, The results from the open-set sentence recognition experiment (Experiment 1) are shown in Table Table C(t) values are displayed in the left panel, and the RT-accuracy C_I(t) values on the right. The dotted lines indicate one standard error (SE) of the mean.Next, the average capacity data for a group of normal-hearing participants are displayed in Figure C_I(t), while the lighter line shows the RT-only C(t). The actual capacity value at a time point is shown on the y-axis and the RT is displayed on the x-axis. To facilitate discrimination between strong integration ability (super-capacity) vs. poorer integration ability (limited capacity), two separate bounds can be found in each panel. First, limited capacity is defined by the dotted line at capacity = ½; the reason is that if processing resources were evenly divided between channels, then we would predict that the energy expended on AV trials would be half of the sum expected from A plus V efficiency  and C(t) at any specific point, along with accuracy, provides information about a participant's processing strategy and integration ability  furnishes information about speed . Also, obtained AV accuracy measured in comparison to the formula, AV) = p(A) + p(V) − p(A) * p(V) furnish information about whether integration is efficient or inefficient in the accuracy domain. Suppose C_I(t) is greater than 1 indicating efficient integration. Further, suppose that C(t) is also greater than 1 and accuracy equals independent race model predictions. This scenario would indicate that the listener is an efficient integrator due to the ability to take advantage of processing time on audiovisual trials rather than accuracy per se. Analogous logic applies to other scenarios in which the listener may show evidence for C_I(t) > 1, but slows down to obtain higher than predicted audiovisual accuracy (C(t) < 1). We shall now discuss capacity results for the individual hearing-impaired listeners by examining C_I(t), C(t), and the discrepancy between predicted and obtained audiovisual accuracy shown in Table Crucially, the difference between This first case involves a 63 year-old male with mild-moderate sudden-onset bilateral high and low-frequency hearing loss of unknown origin. The average pure tone threshold for the low frequency tones (250–1000 Hz) was approximately 20–25 dB HL, while the average threshold for the high frequency tones (2000–8000 Hz) was 45–50 dB HL. This listener wears bilateral hearing aids to facilitate everyday perception, but did not use a hearing aid during testing.C(t) was extremely limited and less than ½ for the preponderance of time points. This finding is intriguing because it also shows that for each point in time, that Participant 1's C(t) was less than the average obtained from the group of normal-hearing volunteers ) was thus exceptionally poor for this listener. This change in speed appears to have resulted from a speed-accuracy tradeoff on visual-only trials since the listener was faster than average, but showed poor accuracy. However, when visual information was combined with the auditory modality, the listener proved capable of taking advantage of the combined auditory-visual information by slowing down to achieve better audiovisual accuracy. When both speed and accuracy were taken into account, Participant 1 displayed evidence for superior integration abilities across most times—particularly slower RTs. This listener elicited greater integration efficiency than normal-hearing participants, and for times greater than 1600 ms post stimulus onset, also demonstrated super-capacity. As observed from the data in Table The upper left hand panel of Figure While this listener had mild low-frequency hearing loss and thus generally poor performance in the auditory domain, he appeared to compensate by slowing down on audiovisual trials relative to visual-only trials in order to maximize accuracy—thus optimizing integration and residual perceptual matching strategies. Significantly, capacity was also reflected by the substantial audiovisual gain on sentence recognition. While auditory-only CUNY sentence recognition was poor due to hearing loss, gain scores were substantially high and only exceeded by Participant 2. This listener's integration skills predict strong face to face communication ability, resulting from the ability to obtain relevant cues from the auditory modality and combine them with visual information. Nonetheless, hearing aids likely facilitate speech communication by providing additional auditory cues, which should help speed-up information processing in difficult listening environments.Case number 2 involved a 22 year old female with bilateral high-frequency bone conductive hearing loss resulting from surgery as a toddler. This listener reported that she never used a hearing aid. The average low frequency threshold was 8 dB, while the average high-frequency threshold was approximately 35 dB HL.C(t) data show evidence for unlimited capacity across most time points, with the exception of very fast processing times. This indicates that capacity was consistent with independent race model predictions since C(t) was approximately unlimited. This finding indicated that Participant 2 was capable of statistically benefiting from visual cues in the RT domain. Furthermore, C(t) was higher compared to the average obtained from the normal-hearing listeners, and also higher than Participant 1's.The panel in Figure C_I(t) shows evidence for unlimited capacity that was driven by RTs. Similar to C(t), C_I(t) was greater than the average obtained from the normal-hearing listeners.One explanation for this particular finding was that the degraded consonant information contributed to slower auditory-only responses and poor auditory-only sentence recognition capabilities; however, enough low-frequency vowel information was present for the listener to enable correct unisensory recognition in the context of a small set size, albeit at a slower pace. When both auditory and visual cues were present, this listener adequately matched the visual to the auditory cues to compensate for the deficits in auditory recognition, and hence, achieved borderline efficient integration. This processing speed difference was perhaps mirrored by the substantial accuracy gain achieved in open-set sentence recognition Table . When coCase number 3 was a 24 year old male with mild to moderate high-frequency sensory neural hearing loss—reportedly due to repeated noise exposure while serving in the military. This listener did not use a hearing aid. His audiogram showed that the average low frequency threshold in the better ear was 15 dB HL, while the average high-frequency threshold was approximately 30 dB HL.C(t) hovers around the lower bound for fixed capacity indicating that this listener is “slow” when extracting visual place of articulation cues and filling in for degraded consonant information in the auditory modality. The results revealed similar auditory-only mean RTs for Participants 2 and 3, although significantly slower audiovisual RTs for Participant 3.Unlike the participant in case 2 who is of similar age and hearing ability, an overview of Participant 3's capacity measures revealed poor integration skills—in part due to poor visual-only skills as reflected in the CUNY sentence recognition task (see Table C_I(t) measure was slightly higher than C(t), although it was lower than 1, and lower than the bound on fixed capacity for his slowest RTs. The reason that the overall integration metric of C_I(t) showed evidence for slightly better integration compared to C(t) was that obtained accuracy levels approximated predicted scores in the speeded word task, making up for sluggish audiovisual RTs. Additionally, this participant showed substantial audiovisual gain on CUNY sentences, indicating the ability to benefit from visual information when combined with auditory speech cues under certain circumstances. Taken together, it appears that this participant has the ability to benefit from multisensory cues although he may fail to benefit in the processing time domain.Crucially, the unitary Case number 4 included a 60 year old male with moderate age related high-frequency hearing loss. This listener was aware of his hearing loss, but did not use a hearing aid to facilitate language perception. His average low frequency threshold in the better ear (right) was 17 dB, while the average high-frequency threshold was recorded as 37 dB.C(t) and C_I(t). This participant showed evidence of unlimited capacity due to the fact that both capacity measures corresponded to independent model predictions for the vast majority of time points. Audiovisual responses appeared slightly faster than race model predictions for early recognition times. Audiovisual mean RTs were faster on average compared to auditory only RTs by approximately 100 ms, and also faster than visual-only RTs, which were quite sluggish. This listener's visual-only perception on both the CUNY sentence perception and speeded word recognition tasks were in the normal range at 13 and 70% correct, respectively; however, the visual-only RTs suggest that he slowed down to achieve this accuracy level. Interestingly, the observation of super-capacity, and that the audiovisual trials were processed faster than auditory-only trials, indicates that visual information about place of articulation sped-up auditory recognition.Similar to the listener from case 2, this listener revealed a strong correspondence between Similar to listener 2, this listener exhibited an integration profile that was consistent with race model predictions. Overall, his integration was superior to the normal-hearing average, which should often be true of listeners with mild to moderate hearing-loss. Despite the relatively poor auditory-only performance due to the loss of high-frequency cues, this listener's integration and lip-reading skills should facilitate face-to-face conversation enough to reduce or eliminate the need for a hearing aid.Case number 5 was a 72 year old woman with a flat audiogram showing evidence for bilateral mild hearing loss. Her average low and high-frequency hearing thresholds in the better ear were measured at approximately 20–25 dB HL. This listener reported being unaware of her hearing loss, and consequently, did not use a hearing aid.Unlike Participant 1's, the capacity results showed slightly limed to unlimited capacity or mildly inefficient integration for responses slower than 1500 ms, but close to unlimited capacity for faster responses. This observation places this listener at odds with the group of normal-hearing listeners who showed super-capacity for faster processing times when comparing this participant's results with those shown in Figure C_I(t) was only marginally less than 1 for the preponderance of recognition times since accuracy made up for the moderate deficits in speed. In fact, since this listener's integration ability approximates the skills of normal-hearing listeners for most time points, she will likely be an effective face-to-face communicator without the use of a hearing aid, except in challenging conversational environments.Overall, the measured The purpose of this study was to provide novel applications of a new capacity approach to identify loci of audiovisual speech integration abilities in hearing-impaired listeners. Specifically, this study extended recent capacity results using single point summaries , and had different hearing loss etiology. In spite of these differences, integration skills as measured by accuracy and capacity were remarkably similar. On the other hand, listeners 1 and 5 were both over 60, and displayed similar audiograms; however, their integration skills differed both qualitatively and quantitatively. The upshot of these findings is that while relationships have been shown to emerge between sensory acuity and integration skills  and C(t) will be necessary. Another important development will involve collecting larger data sets consisting of hearing-impaired listeners with different audiometric configurations and different levels of cognitive functioning. Overall, capacity measures should prove important since audiologists do not comprehensively assess either visual or audiovisual processing capabilities in those suspected of hearing impairment, even though these skills are relevant for face-to-face communication capabilities.Our findings show promise inasmuch as they indicate the importance of incorporating comprehensive speed-accuracy assessment measures. Processing speed is one critical variable predictive of information processing abilities, and one that is well-known to be adversely affected by aging and of course hearing ability. Unfortunately, speed has been overlooked as a viable measure of integration  and C_I(t) using a closed-set speeded word recognition experiment to analyze the extent to which a listener benefits from multisensory cues relative to the predictions of independent models in which integration does not occur. Such a protocol will allow one to determine the locus of a listener's integration capabilities. For example, suppose a listener exhibits high C_I(t) in conjunction with low C(t). This could indicate an inability to benefit from visual speech cues in terms of processing speed, but that slowing down may help one take advantage of visemes to achieve high accuracy.This report provided a basis for a comprehensive methodological approach for examining speech integration in listeners with suspected hearing loss. Accuracy in open-set sentence recognition may be assessed subsequent to traditional audiometric testing. Next, the suggested approach would be to measure Notwithstanding our findings, one may observe that while audiograms and auditory-only hearing ability may be associated with integration Erber, , 2003, iThe 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.


Balaenoptera musculus) and fin  whales also dominate the long-term spectra records in the 15–28 and 89 Hz bands. Blue whale call energy is a maximum during austral summer-fall in the Drake Passage and Bransfield Strait when ambient noise levels are a maximum and sea-ice cover is a minimum. Fin whale vocalizations were also most common during austral summer-early fall months in both the Bransfield Strait and Scotia Sea. The hydrophone data overall do not show sustained anthropogenic sources (ships and airguns), likely due to low coastal traffic and the typically rough weather and sea conditions of the Southern Ocean.Arrays of hydrophones were deployed within the Bransfield Strait and Scotia Sea (Antarctic Peninsula region) from 2005 to 2009 to record ambient ocean sound at frequencies of up to 125 and 500 Hz. Icequakes, which are broadband, short duration signals derived from fracturing of large free-floating icebergs, are a prominent feature of the ocean soundscape. Icequake activity peaks during austral summer and is minimum during winter, likely following freeze-thaw cycles. Iceberg grounding and rapid disintegration also releases significant acoustic energy, equivalent to large-scale geophysical events. Overall ambient sound levels can be as much as ~10–20 dB higher in the open, deep ocean of the Scotia Sea compared to the relatively shallow Bransfield Strait. Noise levels become lowest during the austral winter, as sea-ice cover suppresses wind and wave noise. Ambient noise levels are highest during austral spring and summer, as surface noise, ice cracking and biological activity intensifies. Vocalizations of blue ( The climate of the Antarctic Peninsula is changing in the Southern Hemisphere, with a few degrees Celsius rise in both atmospheric and surface ocean temperatures over the last few decades –3. Assoc2 in area), as it drifted from its origin in the Weddell Sea, grounded within the Bransfield Strait, and broke apart in the Scotia Sea.To further study these ice-related and other sources of natural sound, we deployed arrays of hydrophone moorings in the Bransfield Strait, a protected sea along the western Antarctic Peninsula, and the Scotia Sea, an area of the South Atlantic northeast of the Antarctic Peninsula. A variety of cryogenic sounds were recorded, associated with iceberg grounding and breakup. Icequakes are impulsive broadband signals with durations between 10 and 80 sec and dominant energy over the ~10–500 Hz band. Icequakes may be caused by thermal stresses, as well as physical deformation of the ice from wind, currents, and waves, which can cause fracturing and colliding of large ice blocks and bergs, producing sound . These eOther major components of the ambient sound field near the Antarctic Peninsula are the low-frequency vocalizations of large baleen whales, which dominate the 15–28 and 89 Hz frequency bands  and the In order to monitor the levels and determine the sources of acoustic energy near the Antarctic Peninsula, two arrays of hydrophone instrument moorings were deployed in the Bransfield Strait and Scotia Sea . The BraBalaenoptera musculus) and fin  whales. The pre-amplifier was designed to equalize the spectra against typical ocean noise over the pass-band with an 8-pole anti-aliasing filter. A micro-processor controlled, temperature-correcting crystal oscillator with an average time drift of 1.95 s yr-1 provided accurate timing during the typical 1–2 year deployment duration. The electronics were housed in a titanium pressure case that was attached to a standard oceanographic mooring with anchor, acoustic release, mooring line, and a syntactic foam float to place the sensor at depths of 300–500 m within the ocean water column  because both array locations were primarily designed as passive seismic experiments and set to record low frequency earthquake-generated signals. These sample bands are also ideal for cryogenic sources and for recording the vocalizations of blue  is due to the presence and breakup of the large iceberg A53a in the region (more detail is provided on A53a in the following sections). The breakup of A53a as it sailed past South Georgia Island is the main source of icequakes in the Scotia Sea during the 2007–2008 hydrophone deployment. The second peak in icequake activity during December 2008 is from icequake sources outside of the array, located several array apertures to the north and east of South Georgia. Since A53a had left the area by then, the renewal of icequake activity was likely due to the entry of additional large sea ice into the region.Auto-correlation of icequake activity indicates that the correlation has a maximum value at a lag of 207.3 days, with other peaks at 103.6 and 310.9 days . This isSeveral hundred individual iceberg harmonic tremor (IHT) sounds were recorded on both hydrophone data sets. IHT are seismic and hydroacoustic signals associated with Antarctic icebergs, both adrift and aground, that have only been identified within the last decade ,25. The During April to June 2007, the iceberg A53a (~55 × 20 km) drifted out of the Weddell Sea and through the Bransfield Strait. Although the sounds produced by this iceberg were briefly summarized by Dziak et al. , the conAnalogies to models of tectonic and volcanic tremor suggest three possible IHT source mechanisms: (1) frictional processes, where the iceberg tremors are generated by periodic, discrete stick-slip bursts caused by contact of the moving iceberg with the seafloor or other icebergs , (2) mov2-sec, which is equivalent to the energy release from dozens of individual icequakes and provides an example of the significant contribution that the breakup of these icebergs can have to the ambient sound field.The iceberg then drifted north during July 2007 into the Scotia Sea and began to melt , and witLong-term spectrograms of the acoustic power spectral density and the daily average sound levels recorded on hydrophone M2 in the Bransfield Strait and M4 in the Scotia Sea are shown in Figs From The difference in sound levels between the Scotia Sea and the Bransfield Strait can be more clearly seen in the comparison of sound levels between the two regions in Seafloor earthquakes also contribute to the overall noise spectrum within both the Bransfield Strait  and the The acoustic energy focused in the 15–28 and 89 Hz bands  is from The Drake Passage does not show as large a variation in noise levels as the Bransfield Strait, possibly due to its lack of extensive ice cover. However, the highest noise levels in both the Bransfield Strait and Drake Passage correlate with the times of the highest recorded wind speeds during the austral fall-winter (May–September). This is also consistent with observations of increasing wind speeds leading to increasing noise levels . There iWe next looked at the distribution of blue whale vocalization energy over time  as a meaOverall, hydroacoustic data show that the Bransfield Strait and Scotia Sea are areas of dynamic sea-ice and ice berg activity based on the thousands of icequakes and other cryogenic sound sources recorded in both areas. The icequakes in both regions show a strong seasonal variability, reflecting the freeze-thaw cycle with most icequakes being detected during the austral summer months likely due to increased thermal stress, and are a minimum during early winter . Interesb) earthquakes [Given the large number of icequakes and their high (>190 dB) source levels, the noise from icequakes is clearly a significant and sustained source of ocean sound in the Bransfield Strait and Scotia Sea. For comparison, the maximum icequake source levels (≥220 dB) are equivalent in acoustic energy to moderate to large magnitude . This again is likely due to an increase in sea-ice breakup from seasonal increases in ocean and air temperatures, the presence of A53a in the Scotia in early 2008, contributions from peak blue and fin whale vocalizations during this time, and possibly the interaction of wind with more open water (in the summer) leading to increased wave heights . The dowEven though the seasonal variability observed in the cumulative sound levels is similar for all frequency bands , the 31–Overall, the distributions shown in A closer inspection of the relationship between environmental factors and noise levels  showed tb) ≥ 5 during the hydrophone deployment period as compared to five similar sized earthquakes in the Bransfield Strait (http://earthquake.usgs.gov/earthquakes/search). The lower pace of plate motion in the Bransfield Strait appears to manifest as a large number of smaller magnitude earthquakes, evidenced by the large number of seismo-acoustic signals recorded on the Bransfield back-arc spreading segments during the hydrophone deployments [Seafloor earthquakes also contribute some energy to the overall noise spectrum within both the Bransfield Strait  and the loyments . HoweverAlthough both ships and air gun (for research and/or oil exploration) sounds are occasionally present on the hydrophone data , they arBlue whale sightings are not very common in the Southern Ocean and both blue and fin whale populations are currently at very low levels ,44 folloThe hydrophone data thus re-affirm the value of acoustic monitoring for establishing the presence of the endangered animal populations ,47 in ar