Furthermore, a General Linear Model demonstrated in patients with a prelingual onset of hearing loss enhanced self-evaluated social interactions and activity (NCIQ), when their outcomes were contrasted with those obtained in individuals with postlingual partial deafness (p < 0.05). The study failed to show any effect of collateral tinnitus. Patients not using hearing aids had better audiological performance and, therefore, better sound perception and speech production, as measured with NCIQ. There was no effect of hearing aid use with respect to mental distress. Additional statistically significant correlations seen in patients included those between a steeper slope hearing loss configuration (averaged pure-tone thresholds at 1 and 2 kHz with subtracted threshold at 0.5 kHz) and better audiometric speech detection, between audiometric thresholds and the subjectively rated sound perception (NCIQ), as well as left-ear audiometric word recognition scores and the subjectively perceived ability to recognize advanced sounds (NCIQ). In addition, a longer duration of postlingual deafness, as well as a younger age at the onset were both related to worse speech detection thresholds. The results of the study provide evidence that successful rehabilitation in patients with partial deafness might have to go beyond the standard speech therapy. Enhancement of the regular diagnostic assessment with additional psychological tools is highly recommended. Further investigation is required as to the role of functional residual hearing, hearing aid use and tinnitus, in relation to future outcomes of cochlear implantation.The aim of the study was to evaluate mental distress and health-related quality of life in patients with bilateral partial deafness  before cochlear implantation, with respect to their audiological performance and time of onset of the hearing impairment. Thirty-one patients and 31 normal-hearing individuals were administered the Beck Depression Inventory (BDI), the State-Trait-Anxiety-Inventory (STAI) and the World Health Organization Quality of Life-BREF questionnaire (WHOQOL-BREF). Patients also completed the Nijmegen-Cochlear-Implant-Questionnaire (NCIQ), a tool for evaluation of quality of life related to hearing loss. Patients revealed increased depressive and anxiety symptoms, as well as decreased health-related quality of life , in comparison with their healthy counterparts ( A hearing impairment is not only a disability (a communication dysfunction) but can also be perceived by an individual as a handicap with its psychosocial effects. Patients often encounter confusion, stigmatization or even mockery. The extent of the handicap, however, cannot be predicted from the audiometric profile itself. It has been argued that behavioral and affective variables have to be considered to provide successful management of the disease. Consequently, new tools are being introduced to clinical practice measuring health-related quality of life (HRQoL) in patients, including its core element, mental health, along specific psychological tools to assess psychopathology . Still, however, it remains extremely challenging to capture the non-tangible psychosocial aspects of hearing loss and thereby predict communication and adjustment hardships of patients, as well as their potential benefit from treatment and rehabilitation with e.g. cochlear implantation –5.Whereas patients with a postlingual onset of deafness grow up with a hearing identity to suddenly or progressively be devoid of the auditory sense, those born with a hearing impairment are never exposed to a non-degraded acoustic and speech surrounding. Some authors suggest that underdeveloped communication skills at an early age can deteriorate emotional and social development , with others arguing that an altered identity from hearing to deaf in a later-onset deafness can actually be more detrimental to mental health (see Ohre and colleagues for a review ).Several large- and medium-population studies have indicated increased mental distress among patients with an acquired postlingual hearing impairment (with an onset after developing language skills), as compared to the general population. Depressive/anxiety symptoms and social isolation were found most distinctive –8. FindiIncreased mental distress, and especially elevated anxiety, depression and interpersonal sensitivity have also been detected in the profoundly deaf population using sign language , 11, 12.Partial deafness is a special type of sensorineural hearing loss, with a severe to profound impairment at frequencies above 1–2 kHz and normal to moderately deteriorated hearing acuity at lower frequency bands , 15. Witt(30) = 0.96; p = 0.34]. Nineteen patients were regular users of either one (10 patients) or two optimally fitted hearing devices (9 patients) and 12 patients were non-users (as they had no significant gain). Sixteen patients had chronic non-bothersome bilateral tinnitus. There were 14 patients with a prelingual hearing loss  and 17 patients with a postlingual hearing loss . All patients had well developed verbal skills and used auditory-verbal communication. In all patients audiometric thresholds were measured for frequencies 0.25–8 kHz. PTA values were then calculated for both ears, using averaged thresholds for 0.5, 1 and 2 kHz. Slope was estimated by subtracting the 0.5 kHz threshold from an averaged threshold for 1 and 2 kHz for each ear separately  with a bilateral symmetrical sensorineural hearing loss  participated in the study. Patients were all recruited from among a large pool of patients of the Institute of Physiology and Pathology of Hearing in Warsaw, Poland. Some of the patients were already candidates for cochlear implantation at the time of the study. The mean age of patients was 37.6 ± 7.9 years (M ± SD) (age range 18.5–53.8 years) . Patienlleagues . The out), social relationships , and environment , and includes 26 items in total. A 5-point Likert-type scale is used to provide answers to single questions , STAI-State  and STAI-Trait , indicating more psychopathological symptoms in the clinical population. Furthermore, patients had significantly lower scores on the WHOQOL-BREF scales physical health  and psychological health , which suggested decreased health-related quality of life. All results are presented in Fig. p < 0.05).PD patients obtained significantly higher scores than the normal hearing group in BDI  and the NCIQ social interactions scale . Figure PRE and POST patients obtained comparable mean outcomes in tonal and speech audiometry assessments, as well as hearing aid use and tinnitus  (p < 0.05). Higher PTA values averaged for both ears were associated with lower basic and advanced sound perception scores in NCIQ . The same correlations were found for SDT . In addition, higher SDT averaged for both ears implied lower outcomes on the NCIQ speech production scale. The correlation coefficients revealed for each ear separately were of a similar extent. With a positive correlation apparent for the left-ear WRS and advanced sound perception, there were no significant relationships detected for the right-ear WRS, nor when the scores were averaged for both ears. This was found although WRS scores for the left and the right ear were not statistically different . Moreover, with higher PTA patients had also higher SDT (r = 0.82) and lower WRS (r = −0.82) (averaged for both ears). In addition, lower SDT values were associated with a steeper slope hearing loss (r = −0.53). All these effects were present ipsilaterally and contralaterally. A complementary analysis including only patients with a postlingual onset of partial deafness (N = 17) produced positive correlations between SDT and partial deafness duration (r = 0.53), as well as age at onset (r = −0.56). Variables, age at onset of hearing loss and hearing loss duration, were not found to be associated with any other audiological, demographic or psychological outcomes. There were no statistically significant associations detected for the duration of hearing aid use and duration of tinnitus with aspects of quality of life and psychopathology (p < 0.05).There were statistically significant The current study provides evidence that patients with partial deafness are a specific population that can experience psychological challenges potentially related to their disability. The cross-sectional design, however, does not permit inferring about causal relationships between the sensory loss and the mental well-being. Literature frequently reports an elevated depressed mood, assessed with depression and anxiety questionnaires in the hearing-impaired population , 9, 11. vs bilateral) on any aspect of health/hearing-related quality of life. According to personal communication there were comparable levels of hearing aid satisfaction across all users. Several other studies also failed to show a clear relationship between hearing aid use and mental well-being [The present trial revealed no significant relationship between the objectively measured severity of the hearing deficit, depressive/anxiety levels and HRQoL, replicating outcomes of several cohort studies –3, 7. Thll-being , 3.There were differences demonstrated between patients with a prelingual onset of hearing loss and those who developed partial deafness during their lifetime with respect to social activity and interactions. The prelingual group showed an advantage. The activity domain refers to the subjectively perceived limitations imposed by the hearing loss on daily professional, family and leisure activities, with social interactions assessing the quality of personal contacts with close family and friends, as well as complete strangers. Patients with prelingual partial deafness due to their residual hearing can never become members of the often stigmatized deaf culture using sign language. Furthermore, due to the lifetime experience of disability, they seem to develop efficient communication modes, as well as coping and adjustment mechanisms , 4, 31. As to relationships between various measures applied to patients in the present study, there was a clear correlation detected between audiometric thresholds and the subjectively rated sound perception, suggesting a direct relationship between tests performed in a clinical setting and real-life situations related to hearing problems cf. . FurtherIn the patient group there were also several statistically significant correlations established between various audiological measures. Among other outcomes, it has been found that patients with steeper hearing losses in frequency ranges between 0.5 and 2 kHz found it easier to detect speech (lower SDT scores). This effect of hearing loss configuration on speech perception has already been described by Hornsby and colleagues who argued that patients with ski-sloping sensorineural impairments have better ability to use low-pass filtered speech, when compared to their counterparts with flat hearing loss configurations . ListeniTo be able to extrapolate the findings of the study on the entire population of patients with partial deafness, further longitudinal investigation is required before and after cochlear implantation. In addition, considerable care should be taken to provide a precise account of patient’s self-evaluation of hearing aid use and the collateral tinnitus. The authors are aware that some bias could be introduced to the results by the fact that only patients willing to participate and probably those more extrovert decided to participate in the study and complete questionnaires that directly reflect personal attitudes and feelings.In the current study well-recognized research instruments used in full self-administration served to provide comprehensive information about mental state and health-related quality of life in patients with partial deafness on high frequencies. NCIQ has proven a valuable tool for the pre-CI assessment in this population but its use in the evaluation of treatment outcomes remains to be confirmed in follow-up studies. The results indicate that auditory rehabilitation has to start early and go beyond training speech in laboratory settings. If necessary, the goal of professionals should be to design personalized treatment programs by following individual psychological needs of these patients for longer periods of time, before and after cochlear implantation. Development of new tools is needed to investigate factors that shape the extent to which a hearing loss is considered a handicap and thus what outcomes are to be expected from treatment with a hearing aid or a cochlear implant. Studies involving the population of patients with partial deafness are of particular value given the ever developing medical solutions allowing the preservation of natural hearing, including new systems combining a cochlear implant and a hearing aid, soft cochlear implant electrode arrays, and soft surgical methods , 18. The


Humans differ from other animals in the way they can skilfully and precisely operate or invent tools to facilitate their everyday life. Tools have dominated our home, travel and work environment, becoming an integral step for our motor skills development. What happens when the part of the brain responsible for tool use is damaged in our adult life due to a cerebrovascular accident? How does daily life change when we lose the previously mastered ability to make use of the objects around us? How do patients suffering from compromised tool use cope with food preparation, personal hygiene, grooming, housework, or use of home appliances? In this literature review we present a state of the art for single and multiple tool use research, with a focus on the impact that apraxia (impaired ability to perform tool-based actions) and action disorganization syndrome  have on activities of daily living (ADL). Firstly, we summarize the behavioral studies investigating the impact of apraxia and other comorbidity syndromes, such as neglect or visual extinction, on ADL. We discuss the hallmarks of the compromised tool use in terms of the sequencing of action steps, conceptual errors committed, spatial motor control, and temporal organization of the movement. In addition, we present an up-to-date overview of the neuroimaging and lesion analyses studies that provide an insight into neural correlates of tool use in the human brain and functional changes in the neural organization following a stroke, in the context of ADL. Finally we discuss the current practice in neurorehabilitation of ADL in apraxia and ADS aiming at increasing patients’ independence. Left brain damage caused by ischemic or hemorrhagic stroke is the most frequent neurological correlate of apraxia . HoweverThe purpose of this review is to present a comprehensive summary of the research investigating apraxia syndrome following a cerebrovascular accident (CVA) and its influence on independence during activities of daily living (ADL). First, we provide a systematic overview of the behavioral research investigating impact of apraxia on three basic areas of object and action related abilities: sequencing of action, tool and gesture knowledge and spatiotemporal features of the movement, in the context of basic needs of independence. A particular focus is placed on research investigating the influence of those functions on ADL such as food preparation, personal hygiene, grooming and use of household appliances, or housework tools. The second part of this review is dedicated to the cut-edge neuroimaging research, demonstrating how multi-faceted the neural basis of tool use and ADL is as well as the current state of the art.The most commonly used definition of apraxia was coined by As previously mentioned, apraxia, since the work of Hugo Liepmann, is usually linked to left brain damage . OriginaThe epidemiology of AADS was most recently reported by Several scales are commonly used by the clinical professionals for the assessment of ADL independence in neurological patients. Such scales are usually based on self-report or questionnaire . In the tea making task, one of the patient demonstrated repetitive errors of pouring milk into the teapot rather the mug. Authors referred to it as semantic error, specific for object selection. In principle, ADL can be divided into multiple tool use and single tool use actions , 2013. FA seminal study by This section of the review is organized in a similar fashion to the behavioral part, with division of the studies to the sequencing of subgoals of ADL, then conceptual understanding and finally spatiotemporal features of ADL. To provide an insight into the neural correlates of ADL and apraxia, we present neuroimaging studies with healthy subjects followed by lesion analyses with apraxic patients.p < 0.05 FDR corrected) was used to create the ALE images  concerns the tool manipulation necessary to achieve the intended goal and incorporates spatiotemporal features of the movement. This includes grasping the tool in a correct way and moving it accordingly across space. Functional imaging studies have analyzed the brain areas relevant for selecting the correct grip for tool usage during ADL  or the sFigure 1). Overlaps were found between the different maps for regions processing conceptual and spatial information of tool use and ADL including frontal clusters in the dorsal and ventral premotor areas, in the anterior cingulate cortex, in the parietal lobe along the intraparietal area, the superior parietal lobule, the supramarginal gyrus, around the parieto-occipital sulcus and in the inferior temporal gyrus of the temporal lobe of the left hemisphere. We have found less overlaps in the right hemisphere, which comprise parts of the parietal lobule, precentral gyrus and inferior temporal gyrus. In addition, we report a partial congruency between clusters from sequencing studies and studies focusing on knowledge of tool use. These are associated with activation in the dorsal premotor area, posterior part of the intraparietal area, middle occipital gyrus and fusiform gyrus of the left hemisphere. In summary, ADL and single tool use are complex tasks with multiple aspects to be processed which recruit wide brain networks. Importantly it has to be stated that the neural bases of the three aspects discussed here cannot be clearly separated in actual tool use but need to be integrated to perform ADL. Evidence supporting the importance of the mentioned network is also provided by studies focusing on the neuronal basis of actual tool manipulation, which covered more general or other aspects of tool use  is a system that is currently under development, which is tailored to the needs of AADS patients. The aim is to create fully automatised computer–human interface that provides cues or prompts errors during the performance of ADL . Creating an autonomous system that could aid rehabilitation of ADL in AADS group is a technological and theoretical challenge, which surely will be pursued in the further research developments and projects.Current projects, which aim to provide autonomous systems of guidance for patients struggling with ADL, are primarily tailored for subjects with dementia and use the concepts of domotics (intelligent home environment). One of the projects currently under development is the COACH system, which is designed to provide assistance in hand washing action to residents of nursing home for people with dementia . Based oThe review summarized the most significant research conducted on the impact of AADS on the ADL in stroke survivors. Behavioral, neuroimaging and lesion studies were presented to give an overview of the current state-of-art. Taken together, CVA resulting in lesions in the left or/and right hemisphere has profound consequences on the daily independence of patients during everyday tasks such as food and drink preparation, grooming, personal hygiene, and use of everyday objects. A new approach was adopted to provide a comprehensive description of the unique features of apraxic and action disorganization disorder. The difficulties with execution of ADL were categorized arbitrarily into three components: problems with sequencing of the multi-step actions, conceptual knowledge about tool use and spatiotemporal aspects of the movement. This classification is novel in comparison to the original descriptions of AADS. However, the aim of this approach was to provide a comprehensive insight into the global picture of difficulties CVA patients might experience. Although these themes were presented separately, the evidence suggests those deficits are often intertwined on the behavioral level and also share the neural substrates. In the neural correlates section of this review, the critical role of the left angular gyrus was pinpointed in the sequencing of the multi-step actions. The neural underpinnings of conceptual knowledge were located in the inferior frontal gyrus, the inferior parietal lobe, and middle temporal gyrus. The spatiotemporal features of the execution of the ADL have been linked to the integrity of posterior part of the parietal lobe including the angular gyrus, the parieto-occipital junction and the inferior frontal gyrus. In addition, other areas that were also identified as linked to the ADL performance were discussed, with a conclusion that a wide neural network is involved in cognitive and motor aspects of action planning and execution. In the final section of this review, a strategy training approach was identified as the most efficient and common therapeutic strategy currently used in the rehabilitation of AADS.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.


Etomidate is no longer administered as a continuous infusion for anesthetic maintenance or sedation, because it results in profound and persistent suppression of adrenocortical steroid synthesis with potentially lethal consequences in critically ill patients. We hypothesized that rapidly metabolized soft analogues of etomidate could be developed that do not produce persistent adrenocortical dysfunction even after prolonged continuous infusion. We hope that such agents might also provide more rapid and predictable anesthetic emergence. We have developed the soft etomidate analogue cyclopropyl-methoxycarbonyl etomidate (CPMM). Upon termination of 120-minute continuous infusions, hypnotic and encephalographic recoveries occur in four minutes. The aims of this study were to assess adrenocortical function during and following 120-minute continuous infusion of CPMM and to compare the results with those obtained using etomidate.Dexamethasone-suppressed rats were randomized into an etomidate group, CPMM group, or control group. Rats in the etomidate and CPMM groups received 120-minute continuous infusions of etomidate and CPMM, respectively. Rats in the control group received neither hypnotic. In the first study, adrenocortical function during hypnotic infusion was assessed by administering adrenocorticotropic hormone (ACTH) 90 minutes after the start of the hypnotic infusion and measuring plasma corticosterone concentrations at the end of the infusion 30 minutes later. In the second study, adrenocortical recovery following hypnotic infusion was assessed by administering ACTH every 30 minutes after infusion termination and measuring plasma corticosterone concentrations 30 minutes after each ACTH dose.During hypnotic infusion, ACTH-stimulated serum corticosterone concentrations were significantly lower in the CPMM and etomidate groups than in the control group . After hypnotic infusion, ACTH-stimulated serum corticosterone concentrations recovered to control values within 30 minutes in the CPMM group but remained suppressed relative to those in the control group for more than 3 hours in the etomidate group.Both CPMM and etomidate suppress adrenocortical function during continuous infusion. However, recovery occurs significantly more rapidly following infusion of CPMM. Etomidate is an imidazole-based intravenous sedative-hypnotic Figure . It was Considering the hypothesis that rapidly metabolized soft analogues of etomidate that would not produce persistent adrenocortical suppression could be designed, our laboratory developed methoxycarbonyl etomidate . SimilarSubsequent continuous infusion studies demonstrated that the very rapid metabolism of methoxycarbonyl etomidate, along with its relatively low potency, necessitated very high dosing to maintain hypnosis . This leat libitum.All studies were conducted with the approval of the Institutional Animal Care and Use Committee at Massachusetts General Hospital  and in accordance with its rules and regulations. Adult male Sprague-Dawley rats (250 to 500 g) were purchased from Charles River Laboratories  and housed in the Massachusetts General Hospital Center for Comparative Medicine animal care facility. All intravenous drugs were administered through a femoral venous catheter pre-implanted by the vendor prior to animal delivery to our animal care facility. Each rat was individually caged with controlled temperature (21 ± 2°C) and light-dark cycles (7 a.m. to 7 p.m.) in the Massachusetts General Hospital Center for Comparative Medicine animal care facility, and food and water were provided Etomidate (2 mg/mL in 35% propylene glycol/water) was from Hospira . CPMM was synthesized (> 97% purity) by Aberjona Laboratories  by using our previously described approach  and soluOur goals were to achieve and maintain approximately equivalent anesthetic depths during 120-minute hypnotic infusions. Therefore, for each hypnotic, we used a continuous infusion protocol that, as we previously determined , achieveRats were given intravenous dexamethasone (0.2 mg) to suppress corticosterone synthesis elicited by endogenous ACTH and reduce baseline corticosterone concentrations. Rats were then randomly assigned to CPMM, etomidate, or control groups. Two hours after dexamethasone administration, a baseline blood sample was drawn, a second dose of dexamethasone was given, and the hypnotic infusion protocol was begun. Ninety minutes after the protocol began, ACTH (0.25 mg/kg) was given intravenously to stimulate corticosterone production, and a second blood sample was drawn 30 minutes later . Thus, the corticosterone concentration in the second blood sample was a measure of the extent of adrenocortical inhibition during the final 30 minutes of the infusion Figure .Rats were given intravenous dexamethasone (0.2 mg) and randomly assigned to CPMM, etomidate, or control groups. Two hours after dexamethasone administration, a baseline blood sample was drawn, a second dose of dexamethasone was given, and the hypnotic infusion protocol was begun. After the 120-minute infusion protocol was completed, ACTH (0.25 mg/kg) was given intravenously every 30 minutes for 3 hours to maintain adrenocortical stimulation, and blood samples were drawn 30 minutes after each ACTH dose. The time course of adrenocortical recovery was assessed from the time-dependent corticosterone concentration increase that occurred with repetitive ACTH administration Figure .Serum corticosterone concentrations in blood samples were determined as previously described  by usingP value less than 0.05 was considered statistically significant.All data are presented as mean ± standard deviation. Statistical analyses were carried out by using Prism version 5.0 for the Macintosh . For multiple comparisons, we performed either a one-way or two-way analysis of variance followed by a Bonferroni post-test. A n = 6) and 33 ± 32 ng/mL (n = 6), respectively .Baseline serum corticosterone concentrations prior to the start of the infusion protocol were not significantly different among the three groups and averaged 37 ± 111 ng/mL. After administration of ACTH, serum corticosterone concentrations in the CPMM and etomidate groups were 100 ± 64 ng/mL  or continuous infusion (for anesthetic maintenance) without producing persistent adrenocortical suppression since they are rapidly eliminated. We hoped that such agents might also allow fast emergence from anesthesia, which might ultimately benefit patients while improving operational efficiencies. Our first soft etomidate analogue, methoxycarbonyl etomidate, achieved these goals when given as a single bolus since hypnotic recovery was rapid and adrenocortical suppression was brief . HoweverWith the goals of slowing methoxycarbonyl etomidate's exceptionally fast rate of metabolism and reducing the extent of metabolite accumulation during infusions, we modified the drug's chemical structure by adding a cyclopropyl group directly adjacent to the metabolically labile ester moiety  and shortening the spacer between that ester and the etomidate core Figure . This stWe believe that the introduction of CPMM into clinical practice could have a significant impact on patient care by allowing anesthesia to be maintained, even in older or critically ill patients, without producing significant cardiovascular depression or persistent adrenocortical dysfunction. It may also allow more rapid and predictable anesthetic emergence than is possible with currently available agents.There are several limitations of our study. First is the use of a rat model to study adrenocortical recovery after hypnotic infusion. Rats often metabolize ester-containing drugs (including etomidate) faster than humans ,29. TherThe development of soft etomidate analogues follows a trend in drug development toward agents that are ultra-short-acting and is in response to the need in the operating room and intensive care unit for more precise temporal control of drug effects . EsmololAdrenocortical function was suppressed during 120-minute continuous infusions of CPMM and etomidate in a rat model. However, after infusion termination, adrenocortical recovery occurred in less than 30 minutes with CPMM but persisted for more than 3 hours with etomidate.• Cyclopropyl-methoxycarbonyl etomidate (CPMM) is a rapidly metabolized analogue of etomidate.• CPMM and etomidate suppress adrenocortical steroid synthesis during 120-minute continuous infusions.• Upon termination of 120-minute continuous infusions, adrenocortical recovery occurs faster with CPMM (< 30 minutes) than with etomidate (> 3 hours).• CPMM may be a viable alternative to other sedative hypnotics  in which intraoperative hemodynamic stability and rapid anesthetic recovery are desirable, and adrenocortical suppression persisting for little more than the duration of hypnotic administration is acceptable.ACTH: adrenocorticotropic hormone; CPMM: cyclopropyl-methoxycarbonyl etomidate.Massachusetts General Hospital has submitted patent applications for soft etomidate analogues. Two authors (JFC and DER) and their respective laboratories, departments, and institutions could receive compensation related to the development or sale of the technology presented in this report. DER is a consultant for and holds an equity interest in Annovation BioPharma Inc. , which has licensed this technology for development. RG and EP declare that they have no competing interests.RG participated in the design of the study, carried out the data acquisition and analysis, and participated in drafting the manuscript. EP assisted with study design, data acquisition, and analysis and participated in drafting the manuscript. JFC developed and implemented the computer software to administer hypnotics and assisted with study design and drafting the manuscript. DER conceived and designed the study, carried out the coordination, performed the statistical analyses, and drafted the manuscript. All authors read and approved the final manuscript.


The performance of objective speech and audio quality measures for the prediction of the perceived quality of frequency-compressed speech in hearing aids is investigated in this paper. A number of existing quality measures have been applied to speech signals processed by a hearing aid, which compresses speech spectra along frequency in order to make information contained in higher frequencies audible for listeners with severe high-frequency hearing loss. Quality measures were compared with subjective ratings obtained from normal hearing and hearing impaired children and adults in an earlier study. High correlations were achieved with quality measures computed by quality models that are based on the auditory model of Dau et al., namely, the measure PSM, computed by the quality model PEMO-Q; the measure qc, computed by the quality model proposed by Hansen and Kollmeier; and the linear subcomponent of the HASQI. For the prediction of quality ratings by hearing impaired listeners, extensions of some models incorporating hearing loss were implemented and shown to achieve improved prediction accuracy. Results indicate that these objective quality measures can potentially serve as tools for assisting in initial setting of frequency compression parameters. Frequency lowering techniques are now common in digital hearing aids as an alternative amplification strategy for hearing impaired listeners with severe to profound high frequency hearing loss. For this group of listeners, conventional amplification strategies may result in less than optimal performance due to a combination of inadequate gain at higher frequencies, limited bandwidth of the hearing instruments, and/or the potential presence of high frequency cochlear dead regions Historically, frequency lowering has been achieved in many ways including slow playback, channel vocoding, frequency transposition, and frequency compression, as well as a combination of these alternative strategies Currently, frequency lowering is available as an option in commercial hearing aids as frequency transposition (termed “Audibility Extender”) in Widex hearing aids The differences in frequency lowering implementations can be expected to result in considerably different perceptual effects, even when they are fitted for the same audiometric configuration. For example, McDermott viz. the nonlinear frequency compression (NFC), a brief description of the NFC processing and the evidence surrounding its effectiveness is presented below.Since this paper focuses on a particular frequency lowering strategy, inF is the input frequency in Hz, outF is the corresponding output frequency, cF is the cutoff frequency, p is the compression exponent, and 1/p is the compression ratio (CR). The NFC processing in today’s commercial hearing aids uses compression only for frequencies above a cutoff value. Mathematically, the relationship between input and output frequencies is given bycF ensures that the lower frequency information is unadulterated while the spectral content beyond the cF is compressed into a narrower bandwidth. The NFC scheme has been evaluated with both adults and children. Simpson et al.et al.The specification of the The perceived quality of frequency-compressed sounds will depend on a number of variables, including the parameter settings, hearing loss, the type of sound, as well as highly individual, subjective factors like personal experiences, expectations and preferences. In order to estimate the expected sound quality for a given algorithm setting, sound, and listener, one could either follow a data-driven, i.e. statistical model approach  in general. In contrast to audio codecs, whose aim is to produce output signals that are perceptually indistinguishable from the original input, hearing aids aim to alter the input sound in a way that it compensates for the listeners’ hearing loss. This intentional alternation could result in the processed sound having higher sound quality than the original signal. Therefore, one would need a “perfect” hearing aid to produce the reference signal for sound quality evaluations. Unfortunately, the perfect hearing aid does not exist.However, many hearing aid algorithms can be evaluated with comparison-based quality models. Examples include speech enhancement algorithms that operate on already distorted input signals, like noisy and/or reverberant speech. In these evaluations, the original, clean speech recording can serve as a reference signal, and comparison signals are generated by adding noise or reverberation. Such a comparison-based method may also be applicable in the case of frequency-lowering algorithms. Frequency-lowering will always degrade the naturalness of the input sounds. If subjective sound quality ratings are dominated by the perceived naturalness and not influenced by the possibly improved speech intelligibility or other positive effects of the frequency compression, comparison-based quality models using the unprocessed input signal as a reference appear potentially qualified as predictors of sound quality also for this class of hearing aid algorithms.NFC is a viable choice as an amplification solution for hearing impaired listeners with severe to profound high-frequency hearing loss. Since sound quality is one of the most important factors determining the overall satisfaction and acceptance of hearing aid users Speech signals and subjective quality ratings used to test the quality models were obtained from an earlier study Speech quality ratings were obtained from a group of 12 normal hearing adults , 12 normal hearing children , 12 hearing impaired adults , and 9 hearing impaired children . The listeners with hearing impairment also participated in a field study with NFC hearing aids cF) and compression ratio (CR) parameters, placing this hearing aid in a Brüel and Kjær portable anechoic test box, and recording the output of the hearing aid separately in response to two male speech samples and two female speech samples. A total of five different NFC settings were investigated: (1) cF = 4000 Hz, CR = 2∶1; (2) cF = 3000 Hz, CR = 2∶1; (3) cF = 3000 Hz, CR = 6∶1; (4) cF = 3000 Hz, CR = 10∶1; and (5) cF = 2000 Hz, CR = 2∶1.A database of frequency-compressed speech samples was created to obtain the subjective quality ratings. The database was constructed by first programming a prototype NFC hearing aid with specific cutoff frequency  et al.Parsa This paper evaluated twenty-nine objective quality measures including sub-variants in order to benchmark model performance for prediction of sound quality with NFC processing. The objective models investigated in this paper are considered to be “double-ended” or “intrusive” in their operation. Double-ended models compare features extracted from the signal under test to those extracted from an undistorted reference version of the same signal. The comparisons quantify the degree of perceptual overall difference or similarity. In contrast, single-ended models do not require a reference signal. The models in Examples of objective models in the speech production model parameters include the Itakura-Saito Distance (ISD) A basic objective measure that uses an auditory model is the Weighted Spectral Slope Distance (WSSD) The method for the Perceptual Evaluation of Speech Quality  are computed separately and combined to an overall subjective score predictor overallS in the end. For the Moore quality model, a custom software implementation was developed. Correlations between model output measures and mean subjective quality ratings will be reported for D, nonlinR and overallS.Moore & Glasberg have developed a different loudness model et al.Chen et al.The speech quality model of Hansen & Kollmeier The audio quality model PEMO-Q of Huber and Kollmeier t. The second variant evaluates the temporal course of the instantaneous audio quality, derived from a frame-wise correlation of internal representations, nonlinearly mapped to the overall quality estimator PSMt. In contrast, the PSM is the overall correlation of the complete internal representations. The PSMt is more sensitive to small distortions and more independent of the type of input audio signal PEMO-Q outputs two quality measures: The Perceptual Similarity Measures PSM and PSMBy default, PESQ and PEMO-Q apply an asymmetric weighting of differences between internal representations, putting more weight on “added” or increased elements of the internal representation of the distorted signal than on “missing” or attenuated ones lin, HASQInonlin and HASQIcomb, respectively.Finally, the Hearing Aid Speech Quality Index (HASQI) by Kates and Arehart In summary, the models and measures for speech and audio quality listed in The versions of PEMO-Q and the Hansen model with extensions for hearing impaired data have not been published before and will therefore be discussed in the next section.et al.Modifications of the underlying auditory model of the audio quality prediction methods PEMO-Q and its predecessor, the speech quality model of Hansen and Kollmeier, for describing sensorineural hearing losses have been suggested by Derleth k, which itself is a function of the input signal amplitude I and OHCL. The attenuation and expansion operations are performed instantaneously (i.e. sample-by-sample). For simulating normal hearing, k equals 1 for any I. For simulating impaired hearing, k(I) becomes larger than 1 and increases with OHCL. The input/output function of this stage is shown in In the model stage, the signal amplitude is attenuated by the amount of IHCL. Then, it is raised to the power r and Spearman’s rank correlation coefficient rs. Quality measure values were taken directly as output by the models and not transformed by, e.g., nonlinear regression functions.The performance of the objective measures was characterized by their correlations with averaged subjective quality ratings of all processing conditions (including hidden reference and anchors), quantified by Pearson’s linear correlation coefficient As there were no systematic differences between the quality ratings of normal hearing adults and children, quality ratings were averaged across the complete group of normal hearing subjects. Consequently, results are stated for three groups of subjects separately: (1) normal hearing (adults and children), (2) hearing impaired children, and (3) hearing impaired adults. In addition to averaging across subjects, quality ratings were also averaged across different talkers per processing condition. Results will be stated for both levels of averaging. For clarity, results per subject group will be presented separately in succession and only for a selection of measures. The complete results are reported in the It can be seen from Both subjects and model rate the frequency-compressed signals with the lowest cutoff frequency 2 kHz (condition cr2-fc2k) very poorly compared to the other frequency compression conditions. A possible cause for this non-linear relation between compression cutoff frequency and perceptual effect could be the alteration of speech formant frequencies and/or the interference of down-shifted higher frequency contents with these formants. Vowel formants have significant energy in the second and third formants in approximately the 1000–4000 Hz region for female speech and approximately 750–3000 Hz for male speech, depending on the specific vowel and talker. Recalling that both male and female speech samples were used, it is reasonable to conclude that the cr2-fc2k setting was strong enough to lower significantly the upper formants of many vowels for both male and female speech and was therefore much more noticeable than settings that likely did not disrupt the harmonic relationships within vowels, such as those that used a 4000 Hz cutoff frequency.r up to 0.94), and even higher when averaged across talkers (r up to 0.99). The slightly better performance of the modulation filterbank version of PEMO-Q in the prediction of normal hearing subjects’ ratings (The results obtained for the hearing impaired children are shown in  ratings  was not  ratings .r = 0.99) and a perfect rank correlation (rs = 1) between PSM_lp-B and subjective ratings were achieved when averaged across talkers. Finally, the range of PSM_lp-B (HI) (M16) values is smaller compared to PSM_fb-B (M10) values used for normal hearing subjects, while the range of subjective ratings is about the same in both cases. This difference is due to the greater sensitivity of the modulation filterbank version of the auditory model PEMO used for the computation of PSM_fb-B (M10), compared to the modulation lowpass filter version used for computing PSM_lp-B (HI) (M16).r = 0.88) compared to the results obtained for the children (r = 0.99). In contrast, a very high correlation was achieved by the related quality measure qc-W-B (HI) (M8) (r = 0.96). As described earlier, qc-W-B (HI)  mainly differs from PSM_lp-B (HI)  in the lower cutoff frequency of the peripheral filterbank. Looking at Hearing impaired adults exhibited a different rating behavior than hearing impaired children on average, although there were no significant differences between the distributions of hearing losses in these two groups (children’s hearing losses were somewhat higher than adults’ hearing losses on average). Adults rated NFC conditions generally higher, except for condition cr2-fc2k, whereas their ratings of the anchor condition  were similar to children’s ratings compare . As a reThe obtained results show that predicting the perceived sound quality of nonlinearly frequency-compressed speech by means of objective, perceptual speech/audio quality models is possible. From the models tested, the PEMO-Q (related) measures, in particular, the Perceptual Similarity Measure (PSM) and the speech quality measure qc of the Hansen model (with deactivated frequency weighting function), showed the highest correlations with mean subjective ratings. These measures represent the most “basic” PEMO-based quality measures with the lowest degree of optimization for a specific task and hence the highest generality and robustness. This is in line with results of other studies where different quality measures have been applied for tasks other than those for which they were originally developed e.g. . ThroughA number of objective measures that were previously either applied to speech or audio quality estimation were evaluated in this paper. Of the objective measures based on speech production model parameters (viz. ISD (M26), LAR (M27), LLR (M28)), LAR (M27) performed the best. In the past, these metrics were primarily used for estimating narrowband (0–4 kHz bandwidth) speech quality e.g. . Since sSpeech quality measures  based on the Moore & Glasberg model  performecomb (M19) with subjective ratings increase to 0.83 for normal hearing data , 0.75 (0.78) for hearing impaired adults’ data, and 0.78 (0.82) for hearing impaired children’s data. The HASQI submeasure for linear distortions (M17) achieves very high correlations in this case: 0.93 (0.95), 0.89 (0.95) and 0.95 (0.996) for normal hearing subjects’, hearing impaired adults’ and hearing impaired children’s data, respectively.The Hearing Aid Speech Quality Index  showed good rank correlations and moderate linear correlations with hearing impaired data, but only low correlations with normal hearing data. This is in clear contrast to the results reported in a recent publication on an updated version of the HASQI In the present application, PESQ  surprisingly achieved poor correlations with subjective ratings from normal hearing listeners, but moderate and even high correlations with ratings from hearing impaired adults and children, respectively. The main reason for the poor results regarding normal hearing listeners was found to be a quality overestimation of the lowpass-filtered items by PESQ. Without this processing condition, the linear correlation obtained with the narrow-band version of PESQ increases to 0.89 (0.92) for normal hearing subjects. Correlations obtained with the wideband version of PESQ are lower in this case.et al.The successful “basic” PEMO-Q measures required minimal optimization during development, and use a very simple back-end of the underlying quality model. This result is interpreted as a reflection of the validity of the auditory model originally derived by Dau As already pointed out before, the higher correlation with the adults’ quality ratings obtained with the measure qc-W-B (HI) (M8) compared to PSM_lp-B (HI) (M16) is mainly due to an upward shift of quality predictions for the 2 kHz-lowpass condition and the frequency-compressed condition with the lowest cutoff frequency of 2 kHz, which is in line with the adults’ rating behavior. The upper cutoff frequency of the peripheral filterbank used in the computation of qc-W-B (HI) (M8) is 4 kHz, whereas it is 15.3 kHz for PSM. The two processing conditions of concern cancel (most of) the energy at frequencies higher than 4 kHz. The measure qc-W-B (HI) (M8) does not “notice” the missing energy above 4 kHz, in contrast to PSM_lp-B (HI) (M16). As a consequence, PSM_lp-B (HI) (M16) detects a larger overall difference between original and processed signals which leads to a lower quality estimate. A possible reason for the better correspondence between hearing impaired adults’ ratings and the qc-W-B (HI) (M8) predictions could be a reduced sensitivity of these subjects for missing energy at frequencies above 4 kHz, or/and a lower perceptual weighting of this degradation component when rating the overall quality. Apparently, the hearing impaired children of this study were more sensitive or less tolerant towards the quality degrading effects of frequency compression than the hearing impaired adults. They rated the quality of frequency-compressed speech samples clearly lower than the adults did, although the distributions of hearing losses as described by audiograms are similar in these two groups; the children’s hearing losses are in fact somewhat higher on average than the adults’ hearing losses. There are two factors that may be related to this difference between age groups. First, age-related auditory and/or cognitive declines not revealed by the audiogram, but influencing quality perception might be factors related to this result. Moreover, age-dependent different experiences with and expectations on the quality of sound reproducing systems might play a role and have been speculated before The fact that similar quality rating scores were obtained from normal hearing adults and children does not contradict the hypothesis of an age-related effect, because normal hearing adults in this study were much younger (mean = 24 years) than hearing impaired adults (mean = 69 years). However, in the present data, not all kinds of distortions were rated differently by older listeners, but mainly the frequency-compressed items. Lowpass-filtered and clipped samples received similar quality ratings from children and adults. The same is observed with the quality measure qc-W-B (HI) (M8), in contrast to PSM_lp-B (HI) (M16) for reasons explained above. As a result, qc-W-B (HI) (M8) correlates better with hearing impaired adults’ quality rating scores, whereas PSM_lp-B (HI) (M16) is better suited to predict the hearing impaired children’s ratings. However, the results obtained from hearing impaired adults and corresponding quality predictions obtained with the measure qc-W-B (HI) (M8) might represent a rather special data subset. In contrast, the measure PSM_lp-B (HI) (M16) appears to have a higher degree of generality and is thus recommended for general application.A clear, consistent effect of the kind of modulation processing stage in PEMO-Q on the prediction accuracy could not be found in the results of the present study. Correlations achieved with the modulation lowpass and modulation filterbank versions of the PEMO-Q quality measure PSM, i.e. PSM_lp and PSM_fb, respectively, were mostly comparable and not consistently ranked with regard to subject group, individual vs. averaged talkers, and linear vs. rank correlation coefficients. Since the computational effort required for PSM_fb is about ten times higher than for PSM_lp, the latter might be preferred for practical applications.The first application for which PEMO-Q was originally developed and optimized was the evaluation of audio codecs. For that purpose, a partial, asymmetric assimilation of internal representations of test and reference signals, equivalent to Beerends’ asymmetry weighting of internal representation differences In this paper, we presented results from a study on quality prediction of frequency-compressed speech in hearing aids by different objective speech and audio quality measures. Objective measures based on the audio quality model PEMO-Q and the related speech quality measure qc of the Hansen model achieved good correlations with averaged subjective ratings. The extension of these models incorporated hearing impairment, and improved prediction accuracy for quality ratings from hearing impaired subjects. Hearing impaired adults and children rated the quality of frequency-compressed speech differently. Best prediction results were achieved with different quality measures per subject group; adults’ ratings were predicted better with a model that analyzes a smaller frequency bandwidth. Optimizations of bandwidth and possibly further model parameters were not carried out in the present study, but are considered to bear significant potential for further improvements. The HASQI model was also evaluated, and revealed consistently high results across subject groups for the linear submeasure of this index. Again, further evaluation could reveal optimization to the main HASQI model to further improve its performance.In summary, the present study reports first-of-a-kind results on a broad range of objective sound quality evaluation models of frequency-compressed speech. Further studies are needed to validate the performance of the PEMO-Q quality measures for this application with a larger group of hearing impaired listeners and across more NFC parameter settings. Moreover, the predictability of perceived quality of other sound stimuli than speech, such as music, should be investigated. In addition, the objective modeling of sound quality for frequency lowering schemes other than the NFC scheme evaluated here would require further investigation. In addition, possible influences of age and acclimatization to frequency-compressed sound will have to be investigated and modeled. Research on this topic is driven by the fact that a successful objective speech quality model will have the potential to serve as a valuable supplemental tool for the fitting and evaluation of frequency lowering algorithms in hearing instruments.Appendix S1(DOCX)Click here for additional data file.


The aim of this study is to increase awareness of rare presentations, diagnostic difficulties alongside management of conductive hearing loss and ossicular abnormalities. We report the case of a 13-year-old female reporting progressive left-sided hearing loss and high resolution computed tomography was initially reported as normal. Exploratory tympanotomy revealed an absent stapedius tendon and lack of connection between the stapes superstructure and footplate. The footplate was fixed. Stapedotomy and stapes prosthesis insertion resulted in closure of the air-bone gap by 50 dB. A review of world literature was performed using MedLine. Middle ear ossicular discontinuity can result in significant conductive hearing loss. This can be managed effectively with surgery to help restore hearing. However, some patients may not be suitable or decline surgical intervention and can be managed safely conservatively. Middle ear malformations occur in approximately 1 in 10000 births and can lead to severe conductive hearing loss . PatientA wide range of congenital ossicular abnormalities are described in the literature, including absence of the stapes, stapes suprastructure, stapedius tendon, incus, and oval window alongside fixation of the stapes to the promontory and fallopian canal, as well as various malformations of the malleus, incus, and stapes –9. TheseWe present an extremely rare variant of stapes abnormality that leads to severe conductive hearing loss.SMart piston, Olympus, Southend, UK). Further review of preoperative imaging indicated this ossicular abnormality  mean of 68 dB on the left with normal thresholds on the right. High resolution computed tomography (HRCT) of the temporal bones was initially reported as normal. The patient however elected for exploratory tympanotomy and this demonstrated lack of connection between the stapes suprastructure and the footplate, which was fixed, alongside an absent stapedius tendon . A stapeormality . PostopeThis case demonstrates an extremely rare congenital ear abnormality. Interestingly, this patient presented atypically with progressive rather than nonprogressive conductive hearing loss. Disconnection of the stapes superstructure from the footplate has only been reported once in the literature . AtresiaCongenital middle ear abnormalities can occur independently but often occur in patients with anomalies of the external ear or with craniofacial dysplasia . SyndromWithin the literature, there are differing schools of thought concerning the embryology of the middle ear. It is widely accepted that the ossicles arise from the mesoderm of the first and second branchial arches . However, there is conflicting literature regarding the exact embryological development –19. The Patients with ossicular abnormalities characteristically display a conductive hearing loss. This can be up to 60 dB or maximal, especially in those with oval window atresia . The golThe management of congenital ossicular abnormalities is naturally dependent upon the ossicular malformations present alongside technical and patient factors. Nonsurgical intervention should always be considered. It is generally accepted that in patients with oval window atresia, stapedotomy is the best surgical option  24]. Ex. Ex24]. Congenital ossicular abnormalities are rare causes of conductive hearing loss in childhood and are important in the differential diagnoses for children presenting with nonprogressive hearing loss.Congenital ossicular malformations can often occur in patients with syndromes and a relatively common malformation includes oval window atresia.High resolution computed tomography is the imaging modality of choice and should be reviewed meticulously by experienced clinicians and radiologists.Management options are either conservative or surgical with exploratory tympanotomy, with or without ossicular reconstruction.


Planctomycetes, Verrucomicrobiae, Chlamydiae, Lentisphaera, Poribacteria, OP3, WWE2) presenting different lifestyles, cell plans and environments. Planctomyces and several Verrucomicrobiae exhibit a complex cell plan, with an intracytoplasmic membrane inducing the compartmentalization of the cytoplasm into two regions (pirellulosome and paryphoplasm). The evolution and function of this cell plan is still subject to debate. In this work, we hypothesized that it could play a role in protection of the bacterial DNA, especially against Horizontal Genes Transfers (HGT). Therefore, 64 bacterial genomes belonging to seven different phyla (whose four PVC phyla) were studied. We reconstructed the evolution of the cell plan as precisely as possible, thanks to information obtained by bibliographic study and electronic microscopy. We used a strategy based on comparative phylogenomic in order to determine the part occupied by the horizontal transfers for each studied genomes.The PVC super-phylum gathers bacteria from seven phyla (Simkania negevensis (Chlamydiae) and Coraliomargarita akajimensis (Verrucomicrobiae), whose cell plan were unknown before, are compartmentalized, as we can see on the micrographies. This is one of the first indication of the presence of an intracytoplasmic membrane in a Chlamydiae. The proportion of HGT does not seems to be related to the cell plan of bacteria, suggesting that compartmentalization does not induce a protection of bacterial DNA against HGT. Conversely, lifestyle of bacteria seems to impact the ability of bacteria to exchange genes.Our results show that the bacteria Our study allows a best reconstruction of the evolution of intracytoplasmic membrane, but this structure seems to have no impact on HGT occurrences.This article was reviewed by Mircea Podar and Olivier Tenaillon.The online version of this article (doi:10.1186/s13062-016-0144-3) contains supplementary material, which is available to authorized users. Rece3, WWE2) . This id3, WWE2) . The phy3, WWE2) , 13. PVC3, WWE2) , 16–21.Planctomycetes are implicated in carbon and nitrogen cycles [Chlamydiae are pathogens of mammals [Chlamydiae and Verrucomicrobiae are symbionts [Verrucomicrobiae and Planctomycetes [Planctomycetes and Chlamydiae [Planctomycetes. Among these characteristics, we were specifically interested in the compartmentalization of bacteria. This feature is characterized by a specific cell plan, and concerns all the Planctomycetes [Verrucomicrobiae [Lentisphaera and one Poribacteria [Candidatus Kuenenia Stuttgartiensis [Gemmata obscuriglobus [Eukaryota [The bacteria of this super-phylum show different interesting characteristics , 22. Somn cycles , 24 or sn cycles , many Ch mammals , 27, somymbionts . But PVComycetes , 29, abslamydiae , 31, cralamydiae  in Plancomycetes , 34, somicrobiae , one Lenbacteria . An intrbacteria ), and thbacteria , 33 presbacteria , 38. Somrtiensis , 39, 40,rtiensis  in the srtiensis  in Gemmariglobus . Compartukaryota , this obukaryota –48. 2- Aukaryota . 3- Someukaryota .Considering the reality of the existence of a compartmentalization in PVC bacteria, it would be interesting to determine the function of this specific cell plan; indeed, no previous studies had been able to determine it. Here we assumed that this membrane could be a protection against Horizontal Gene Transfers (HGT). This hypothesis was based on observations of the impact of different membranes already studied : the role of the internal membrane in Eukaryotes, or the influence of host membrane on genomes from intracellular bacteria (which limits the contact between foreign elements and DNA). We used a phylogenomic strategy of HGT detection to reconstruct the history of HGT during the evolution of PVC bacteria, before and after appearance and disappearance of compartmentalization.Verrucomicrobiae, Planctomycetes and Lentisphaerae) and eight non compartmentalized bacteria in one phyla (Chlamydiae). These bacteria were used in our study, but we also selected seven bacteria with an unknown cell plan, distributed in two phyla (Verrucomicrobiae and Chlamydiae). These data allowed the reconstruction of cell plan evolution with a good reliability, however it was still difficult to conclude about ancestral state at different nodes of the tree, especially in Chlamydiae phylum . It was interesting to add new information in the dataset, in order to improve this reconstruction.PVC bacteria present two different cell plan, compartmentalized by an intra cytoplasmic membrane (ICM) or non compartmentalized. They also present two different lifestyles: allopatric lifestyle, for bacteria isolated from other microorganisms (obligate intracellular bacteria living in non amoeba cells) and sympatric, for bacteria in contact with other microorganisms. The studies already published allowed determining cell plan of 26 PVC bacteria: PVC super phylum presented 18 compartmentalized species distributed in three phyla . The ancestral reconstruction permitted to date the appearance of intracytoplasmic membrane to the root of PVC super-phylum with a probability of 99.8 % (against 96.1 % without our new data). The eight non compartmentalized Chlamydiae form a monophyletic group and represents all the allopatric bacteria of the super-phylum, their ancestor was supposed to be allopatric and was predicted as non compartmentalized, suggesting a disappearance of compartmentalization in this ancestor (with a probability of 99.4 %).These microscopic observations revealed cell plan information about two bacteria, whose a ion Fig. . The comp-value = 3.0*10-4). In species tree, an important decrease of HGT proportions was identified after the conversion of Chlamydiae to intracellular allopatric lifestyle. Among sympatric bacteria, we could not identify a significant difference of HGT proportion according cell plan, regardless the type of HGT studied  . These observations suggest that intracytoplasmic membrane has no impact on HGT frequency.HGT were detected by phylogenomic methods, the position of HGT in species tree allows to differentiate HGT occurred only in modern species (specific HGT) and HGT occurred in their ancestors (non specific HGT). We identified 27275.0 proteins acquired by HGT . The proteomes of sympatric compartmentalized bacteria present 13040.0 HGT with 7259.0 specific HGT (6.2 % of proteomes) and 5781.0 non specific (4.9 % of proteomes). The proteomes of sympatric non compartmentalized bacteria contain 12686.0 HGT, whom 5466.0 specific (6.7 % of proteomes) and 7220.0 non specific (8.9 % of proteomes). Allopatric non compartmentalized bacteria present 1549 proteins acquired by HGT in their proteomes, with 160 specific HGT (2.0 % of proteomes) and 1389 non specific HGT (17.7 %). When we compared their HGT proportions . Conjugation genes are significantly over represented in the genomes of Intracellular allopatric bacteria, and underrepresented in extracellular compartmentalized bacteria .The compartmentalization of bacteria could disturb only some particular mechanisms of HGT processes, leading to a low impact on HGT global proportions. The study of mobilome revealed that no element present proportions varying significantly according cell plan . The micrography of C.akajimensis completes the studies already realized on the Verrucomicrobiae and reinforces the probability of compartmentalization in the last common ancestor of Verrucomicrobiae . These observations allowed to conclude with fewer uncertainties our study concerning the HGT in compartmentalized bacteria.The results of electronic microscopy allowed to defined  in 2010  and 2013 in 2010  respecti in 2010 . The dif in 2013  and Kuo- in 2013 . These mCell plan evolution reconstruction indicated that compartmentalization appeared at the root of PVC super-phylum and disappeared once, in the group of allopatric intracellular bacteria. This disappearance seems to occurred simultaneously with bacteria conversion to intracellular allopatric lifestyle. Intracellular allopatric bacteria are isolated from other bacteria, so their genomes are protected against HGT. Conversely, extracellular and intracellular sympatric bacteria are more exposed to exchanges because they are in contact with many microorganisms. An hypothesis could be that the intracytoplasmic membrane plays a role in protection of genomes against HGT, as host membrane of intracellular bacteria or eukaryotic membrane . This inChlamydiae after their conversion to the intracellular allopatric lifestyle is probably due to the physical isolation from other microorganisms, which prevents the opportunities of HGT [Chlamydiae present an important rate of ancient HGT with eukaryotes, significantly higher than other bacteria . This observation is consistent with the existing literature dedicated to transfers between plants and Chlamydiae [Chlamydiae in chloroplast endosymbiosis; The HGT proportions detected in two Planctomycetes (Planctomyces maris and Candidatus Kuenenia stuttgartiensis) correspond to these observed by Kamneva et al in 2012 [Spirochaetes seem to exchanges more with Firmicutes as it was already detected in previous studies [The analysis of HGT in the different groups of bacteria highlighted the role of lifestyle on HGT process and the absence of impact of compartmentalization. The important decrease of HGT proportions observed in s of HGT . This ags of HGT , 54 withs of HGT , 56. Thelamydiae , 58 that studies , 60.A similar HGT proportion in compartmentalized and non-compartmentalized bacteria does not necessary contradict the hypothesis of protection against HGT by compartmentalization. Indeed, the intracytoplasmic membrane of PVC bacteria can represents a barrier to HGT by disturbing only some transfers mechanisms mediated by mobilome elements. This perturbation could lead or not to a decrease of HGT level, depending if the deficiency of some mechanisms are offset by the others. The absence of difference in mobilome elements quantities and proportions between the different groups of bacteria indicated that the compartmentalization has no impact, not only on HGT proportions, but also on elements implicated in HGT process. The lifestyle seems to have an influence on two mobilomes elements, proportions of transposases and conjugation genes. The high proportion of transposases in intracellular allopatric bacteria could be related to the importance of transposable elements in genomes of intracellular bacteria , 62. HowThe function of compartmentalization in PVC bacteria remains unknown and the causes of intracytoplasmic membrane disappearance in intracellular isolated bacteria is still unresolved.Verrucomicrobiae and Chlamydiae allow a best definition of ancestral and modern states of compartmentalization and so, permit to reconstruct intracytoplasmic membrane evolution in PVC bacteria. But this compartmentalization seems have no significant impact on HGT quantity, HGT proportion or partners of transfers.The different pictures obtained for Planctomycetes, Verrucomicrobia, Lentisphaerae, and Chlamydiae. Bacteria from three phylogenetically close phyla were chosen as negative controls, based on a reference tree [Bacteroidetes, Spirochaetes and Chlorobi. We retrieved the proteomes of all bacteria selected in genomes NCBI database  was grown within a culture of its amoebal host A. castellanii. At H6, and H16 post-infection, cultures were centrifuged at 2000 rpm/min during 10 min, and the pellets were fixed for electron microscopy in a fixative solution . The second species, Coraliomargarita akajimensis  was cultivated directly into Bacto Marine Broth medium (Difco). Growth occurred after 6 days of cultivation, and when the bacterial suspension reached the exponential growth phase, it was centrifuged at 5500 rpm/min during 30 min, and fixed for electron microscopy in the same fixative solution. An aliquot of each culture was kept for DNA extraction, followed by a standard 16S rRNA PCR and sequencing, in order to confirm the bacterial identification. For both bacteria, Electron microscopic observations were realized in different steps: for embedding, cells were fixed for 1 h with glutaraldehyde 2.5 % in 0.1 M sodium cacodylate buffer and washed three times. Cells were post-fixed for 1 h with 1 % OsO4 diluted in 0.2 M Potassium hexa-cyanoferrate (III) / 0.1 M sodium cacodylate solution. After four 5 min washes with distilled water, cells were gradually dehydrated with ethanol by successive 10 min baths in 30, 50, 70, 96, 100 and 100 % ethanol. Substitution was achieved by successively placing the cells in 25, 50 and 75 % Epon solutions for 15 min. Cells were placed for 1 h in 100 % Epon solution and in fresh Epon 100 % over-night under vacuum at room-temperature. Polymerization took place with cells in fresh 100 % Epon for 24 h at 60°. Ultrathin 70 nm sections were cut with a UC7 ultramicrotome (Leica) and placed on HR25 300 Mesh Copper/Rhodium grids . Electron micrographs were obtained on a Tecnai G20 TEM operated at 200 keV equipped with a 4096 × 4096 pixels resolution Eagle camera (FEI)).Cell plans of bacteria are determined thanks to transmission electron microscopy pictures already available in bibliography , 34, 35 Bacteroidetes-Chlorobi-Spirochaetes thanks to 12 markers common to the 65 species , two elongation factors (Tu and Ts), two DNA polymerase subunits, CTP synthetase, and four tRNA ligases). The selected sequences were concatenated and Mega5 [We reconstructed the species tree of PVC bacteria and nd Mega5  softwarens 16S, 2S, 30S, tThanks to information about compartmentalization in bacteria, we reconstructed the ancestral state of cell plan with parsimony method , conjugation genes and plasmids, these three elements are involved in entrance of foreign sequences in bacteria; Transposases, integrases and CRISP (candidate or confirmed), regulating positively or negatively the sequences integration in recipient genome; and tRNA used for sequences translation. Different databases were used as RepBase  and CRISde novo (ORFans). Blast against nr database allowed the identification genes presenting no identifiable homologous genes , considered as ORFans.OrthoMCL  was usedAkkermansia muciniphila (Verrucomicrobiae), all sequences in Blast results derived from Verrucomicrobiae were removed). Sequences with e-value > 10-5, coverage < 60 % or identities < 30 % were also removed. Then, species of the first ten sequences remaining in Blast result were identified. If species identified do not belong to one of the sister phyla studied, gene gain is probably an HGT . The position on gains in species tree allows to differentiate HGT occurred only in modern species (specific HGT) and HGT occurred in the ancestor (non specific HGT). We calculated the quantity of HGT and the percentage of proteins present in each species, due to transfers. For each HGT detected (recent or ancient), we determined with which organism the transfers were realized.Phylopattern  pipelineFor each elements studied , we performed a comparison of variances between the different groups of bacteria (based on cell plan). We tested the normality of data distribution by the shapiro-Wilk test and the homogeneity of variance by Levene test. These tests were followed by a comparison of variance between the different classes, in order to evaluate if there was a significant difference between them . Tests of Nemenuyi  or Tukey  were performed to obtain a comparison of each pairs of classes. We realized also these statistical analysis with bacterial groups based on the phyla, to determine if classes based on phyla present the same results that classes based on cell plan. If it had been the case, it would have been impossible to determine if differences observed were related to cell plan or to phylogenetic relations.“In their study, Pinos et al tested their hypothesis that cellular compartmentalization, observed in some representatives of the PVC super phylum, is correlated to the frequency of horizontal gene transfer. The authors performed ultrastructural analyses of a representative from the Chlamydia and one from Verrucomicrobiae, which were shown to be compartmentalized. That information was combined with results from literature on other PVC species and was correlated with phylogenomic analyses on HGT frequencies, based on completed genomes. The result indicates that compartmentalization is in fact not significantly impacting HGT, which has a stronger correlation with the organismal life-style.The study is interesting and original, even though the conclusion did not support the original hypothesis. It does provide a foundation for additional tests and focused study of related organisms/genomes, which may increase the signal to link compartmentalization to more subtle evolutionary genomic effects.”“In the manuscript entitled “Compartmentalization in PVC super-phylum: evolution and impact”, Sandrine Pinos and colleagues use both microscopy and a phylogenetic approach to study Compartmentalization and its potential impact on horizontal gene transfer.The idea is interesting, but the paper is not very clear in its present form. The usage of microscopy is not introduced properly, statistics and methods are over-simplified and impossible to reproduce. I would consider therefore that the paper needs very substantial rewriting and the phylogenetic analysis should include uncertainty in the phylogeny.”Reviewer 1 (Mircea Podar): The manuscript needs extensive work in correcting the many language errors . It also does not provide sufficient detail to evaluate the power of the phylogenomic analyses that were performed. While I like the concept of this study and the overall approach, the manuscript requires a complete overhaul.Reviewer 2 (Olivier Tenaillon): How robust is the tree? Before being able to conclude on the ancestry of a trait, the robustness of the phylogeny has to be tested. In my experience a 16S phylogeny is not very robust and much more loci should be involved in the reconstruction, bootstrap values should be presented. Character reconstruction should also take into account the tree uncertainty. This is critical for both figures and both character mapping procedures.Reviewer 2 (Olivier Tenaillon): line 219: “If species identified do not belong to one of the close phyla studied, gene gain is probably an HGT” how close is close, the threshold use should be clearly defined?”The different comments and questions concerning our phylogenomic strategy indicate an important problem in our presentation of this method and its results. In order to correct these problems, we expanded the methodology part of phylogenomic strategy, by adding indications and some examples . We also added, in the Additional file 3, phylogenies of PVC bacteria and Bacteroidetes-Spirochaetes-Chlorobi, with the indication of bootstrap values. These phylogenies are more robust than those of the first version, due to the use of ten supplementary markers, for alignment and phylogenetic reconstruction. We want to underline that some results obtained by our phylogenomic method are convergent with results already observed in previous studies (line 155–161), this is an indication of the relative efficiency of our method.Author’s response: The improvement of the phylogeny allowed a better reconstruction of cell plan evolution in PVC bacteria. However, In order to take into account the problem of phylogenetic uncertainties, we used another method of ancestral reconstruction that not only considers these uncertainties, but also provides the probabilities of each state of cell plan at each nodes of the tree. The results of this analysis were included in the Figure 2, they led to a better vision of PVC bacteria evolution and highlighted the importance of microscopic data.Reviewer 2 (Olivier Tenaillon): Introduction - The link between horizontal gene transfer and compartmentalization is not clearly stated in the introduction. Two arguments are mentioned, the second one a physical limit is somehow simple to understand, but the first one remains enigmatic: “the presence or absence of compartmentalization in the different PVC bacteria and the reconstruction of ancestral states”. What is meant by this sentence is not clear to me. I think developing more on the subject would improve the introduction.This sentence is difficult to explain without the knowledge about the compartmentalization and the lifestyle of PVC bacteria, however, it could be long to detail all these information in introduction. Consequently, we decided to remove this sentence from the introduction, we presented and explained it in the results part. This sentence resumed the idea that if Chlamydiae are the only non compartmentalized PVC bacteria sequenced and also the only intracellular bacteria of this super-phylum, if the compartmentalization protects genomes against HGT, its lost in the intracellular species could be related to the isolation of these bacteria that induces a low proportion of HGT, and leads to the uselessness of compartmentalization. So, the bibliographic knowledge about the presence or absence of compartmentalization in species suggests the hypothesis of a role of the ICM in protection against HGT.Author’s response: Reviewer 2 (Olivier Tenaillon): Results - The result section starts with the description of microscopic structures. It is not introduced in anyway. The two first paragraphs should be inverted: first mention that the compartmentalization is unknown in some species, then present the evidence for the ones lacking information.Discussion - again no clear explanation of the interest of the microscopy.As the result of the comments of reviewers, we decided to reorganize the results of microscopic analyzes to improve our presentation. We insisted on the importance of these data in our study by adding explanations of their use in ancestral reconstruction (71–80) and their role in the study of the relation between HGT and compartmentalization (97–99). We realized a more detailed ancestral state reconstruction, with a more complex method  that provided the probabilities of each cell plan states at each nodes of the tree. These analyzes highlighted the importance to add our data to the study, indeed our supplement of information allowed to improve significantly the probability of compartmentalization for several nodes of tree.Author’s response: Reviewer 2 (Olivier Tenaillon): Results - The term allopatric and sympatric should clearly be defined in the present context and not in the method. Being an evolutionary biologist rather than a microbiologist, I do not support the use of these terms that refer to speciation and not to accessibility of foreign DNA, but as the author have previously used them… It should nevertheless be explicitly defined, with the present definition it is not clear who has access to other microbes.Indeed the terms allopatry and sympatry needed to be clearly defined, we added the complete definition in methodology part and we also put a short definition in the results part.Author’s response: Reviewer 2 (Olivier Tenaillon): The mobilome analysis are not described enough to be reproducible.We improved the paragraphs dedicated to the mobilome analysis by adding explanations about our strategy . We completed these explanations by a table of the databases used to retrieve the mobilome elements, available in the Additional file 4.Author’s response: Reviewer 2 (Olivier Tenaillon): “line 206 “Genes absent to all orthologous groups are either acquired by HT or generated de novo”: this sentence is not clear. Sometimes HT is used sometimes HGT.The term HT indicates an horizontal transfer of sequence, this sequence could contain one or more genes (average of genes implicated by transfer is 1.5). The term HGT refers to a transfer of one gene. As the use of these two terms complicated our manuscript, we decided to use only the term of HGT, and we adapted our data in consequence.Author’s response: Reviewer 2 (Olivier Tenaillon): line 206 NR database: the author should say what it is and use either nr or NR.Indeed we used the nr database, we corrected this mistakes in manuscript.Author’s response: Reviewer 2 (Olivier Tenaillon): Figure - species names should be written in full and in italic with no underscore. Line 70–75 Statistics should be given to compare “sympatric” groups.The figures were corrected and we added the statistics for sympatric bacteria comparison in the paragraph.Author’s response:


Individuals with the CDKL5 disorder have been described as having severely impaired development. A few individuals have been reported having attained more milestones including walking and running. Our aim was to investigate variation in attainment of developmental milestones and associations with underlying genotype.CDKL5 mutation and information on early development. Kaplan-Meier time-to-event analyses investigated the occurrence of developmental milestones. Mutations were grouped by their structural/functional consequence, and Cox regression was used to investigate the relationship between genotype and milestone attainment.Data was sourced from the International CDKL5 Disorder Database, and individuals were included if they had a pathogenic or probably pathogenic The study included 109 females and 18 males. By 5 years of age, only 75% of the females had attained independent sitting and 25% independent walking whilst a quarter of the males could sit independently by 1 year 3 months. Only one boy could walk independently. No clear relationship between mutation group and milestone attainment was present, although females with a late truncating mutation attained the most milestones.Attainment of developmental milestones is severely impaired in the CDKL5 disorder, with the majority who did attain skills attaining them at a late age. It appears as though males are more severely impaired than the females. Larger studies are needed to further investigate the role of genotype on clinical variability. CDKL5) gene has been limited to case studies and small case series (n = 20)[CDKL5 mutation met the clinical criteria for the early-onset seizure variant of RTT, which specifically requires a period of developmental regression[CDKL5 gene.The majority of research on individuals with mutations in the cyclin-dependent kinase-like 5 ((n = 20)–3. These(n = 20), we founMECP2)[The literature suggests that individuals with the CDKL5 disorder rarely experience normal development and that the majority have refractory epilepsy with onset within the first few months of life and severe global developmental delay–8. ThereMECP2)–15. For CDKL5 mutation was considered to be pathogenic or probably pathogenic, and the caregiver had completed the questionnaire section on early development. Due to the heterogeneity of mutations within our sample, each individual’s mutation was grouped according to its predicted structural/functional consequence[Information on individuals with the CDKL5 disorder was obtained from the newly developed International CDKL5 Disorder Database, which was established in September 2012. Data is collected from caregivers in the form of online or paper-based questionnaires. Caregivers were asked to refer to their child’s infant health record, diaries or available medical records when completing the questionnaire. Cases were included in this study if the individuals sequence. These gTo investigate early development, we included information on the age of attainment of the following major developmental milestones: sitting, standing and walking independently; raking grasp and pincer grip; and babble and use of single words. The timing of attainment of these milestones was investigated using Kaplan-Meier time-to-event analysis. This allows an appropriate estimate of the proportion of individuals who will attain a skill by a particular age whilst taking into account those who are still yet to attain the skill. To investigate the relationship between mutation type and the attainment of these particular milestones, separate time-to-event analyses by mutation group were undertaken and the median age and proportion that attained each skill determined. Cox regression was used to investigate the relationship between each skill and mutation group.Information on the attainment of additional milestones, for which we did not collect an age of attainment, was also included. For these skills, individuals were grouped according to their current age at time questionnaire completion  and we determined the proportion of each age group who had attained the milestone of interest. These age groups were chosen based on the expected developmental level of the children. All the milestones included in our study (excluding the use of phrases) would have been achieved by 1.5 years of age in a normally developing child. We therefore grouped those aged 1.5 years and under as it was likely that these children were yet to attain many of these skills. This allowed us to account for young individuals who had not yet had the opportunity to attain these skills.Approval for this study was provided by the University of Western Australia Human Research Ethics Committee, Perth, Western Australia, Australia (RA/4/1/5024).As of May 2014, there were 127 individuals for whom information on early development had been provided. Of these, 109 (86%) were female  and 18 (14%) were male . The distribution of mutations within our sample is shown in Table n = 27/105), and a quarter was able to stand independently by 5 years (range 10 months to 6 years 11 months). Independent walking was attained by 22% of females (n = 24/109), and time-to-event analysis showed that a quarter had attained independent walking by 4 and a half years (range 1 year 6 months to 6 years 5 months). For the 18 boys in our study, information on the age of attaining sitting was known for all but one, of whom six could sit independently with a quarter attaining independent sitting by 1 year 3 months (range 8 months to 3 years)  had attained independent sitting. The median age at attainment of independent sitting was 3 years (range 6 months to 5 years) with almost three quarters achieving unaided sitting by 5 years of age Figure . Indepenn = 14/106) of whom 10% had attained a pincer grip by 3 years (range 1 to 12 years). For the males, information was available for all but one on the age of attaining a raking grasp. Ten percent of the males attained a raking grasp by 1 year 10 months and one male attained a pincer grasp at 2 years of age  had attained a raking grasp at a median age of 5 years (range 5 months to 12 years) , and 10% would attained single words by 1 year 6 months and just under a quarter by 7 years of age (range 9 months to 7 years). Of the males in our study, information on age of attaining babble was available for all but three (n = 15) of whom five attained the ability to babble. A quarter attained babble by 7 months of age (range 6 months to 1 year 8 months)  had attained babble. A quarter of whom achieved babble by 1 year of age (range 3 months to 6 years) and just under a half by 6 years Figure . Single  Figure . There wThe proportion of females and males who attained other gross motor, fine motor and communication skills not included in the time-to-event analysis is shown in Table p value 0.026, 95% CI 1.11–5.36).A higher proportion of females with a truncating mutation after aa 781 attained gross motor, hand function and communication milestones than the other groups Table . AlthougThe acquisition of developmental milestones was severely impaired in our sample of individuals with the CDKL5 disorder. This was especially true for males, with nearly half not achieving any of the major gross motor, communication or hand function milestones investigated. There was variability, with a few girls eventually attaining most if not all of the milestones we investigated. If milestones were attained, they were significantly delayed. Despite small numbers, it appears as though mutation type may play some role in this variation, with girls with a truncating mutation after aa 781 attaining more milestones than those who had a mutation resulting in no functional CDKL5 protein.CDKL5 gene. With advances in genetic technologies, less severely affected children are being tested and identified, thus increasing the variability seen initially in children with the CDKL5 disorder. Regardless, it is important that this variability in functional ability be recognised both by clinicians and researchers.Our study has shown that there is variability in the attainment of developmental milestones within the CDKL5 disorder. The majority of females did achieve some milestones, most frequently involving gross motor skills. For most, these milestones were attained at an older age than expected for the general population. Using tCDKL5 mutations. Advances in genetic technology for diagnostic purposes mean those children who would previously never have been identified, such as our young boy who could run and who had never been diagnosed with epilepsy, are being diagnosed. These findings suggest that a different spectrum of boys with the CDKL5 disorder, who until now have not been diagnosed, may exist.In our study, the males with the CDKL5 disorder had more impaired development than the females. Again, there was variability, with some of the males having less impaired development. Half of the boys in our study had attained rolling, a quarter learned to sit independently by 1 year 3 months and a quarter used babble by 7 months . There were four boys in our study who had attained more developmental milestones. The first was a 10-month old who could sit independently, babble and transfer objects from hand to hand . The second was a 2 and a half year old who could stand independently, transfer objects from hand to hand and babbled . The third was a 3 and a half year old who could sit independently, had limited hand use and could use single words . The fourth was a 4 and a half year old who had never had epilepsy, could walk independently (even run) and had a pincer grip but had language impairment (babble only) . There have been few boys with the CDKL5 disorder described in the literature, 32–38. Our understanding of the functional role of CDKL5 is still limited, but CDKL5 has been shown to be involved in neuronal cell differentiation and proliferation, dendritic arborisation and dendritic spine morphology–41. It iCDKL5 gene. Earlier research suggested that loss of the C-terminal region can in fact act as a loss of function mutation due to loss of localisation ability and increased self-phosphorylation (increased capacity to phosphorylate the kinase itself)[The relationship between genotype and phenotype in the CDKL5 disorder has only been specifically investigated in one other study. This st itself), 44. One itself). No cleaOur study is the first major international data collection of individuals with the CDKL5 disorder. This gives us the advantage of having the largest data collection on individuals with the CDKL5 disorder. Our data collection tools were also designed to capture clinical information specific to the CDKL5 disorder, not other disorders such as RTT. It is apparent that there is much variability within this disorder, and therefore larger numbers are still needed to provide a more comprehensive representation of the clinical presentation. Recall error, especially for caregivers with older children, is likely. We have limited this by asking caregivers to refer to their child’s medical records and diaries before completing the questionnaire. Because these children exhibit symptoms from an early age, developmental gains are most likely to be well recorded, as caregivers are paying special attention to their child’s development. We found that many parents knew whether their child attained a particular milestone, but the age that these skills were attained was not always known, which is demonstrated by the missing data in our study. Changes in diagnostic technology and increased awareness of the CDKL5 disorder mean that children are being diagnosed younger, and a more variable spectrum is being identified. This results in the ‘cohort effect’ to which we have alluded, where the younger are less severe than previously diagnosed children. The identification of younger children means that this data collection will have the capacity to collect prospective data on developmental milestones. Finally, our study will only reach caregivers of individuals who have access to the internet and therefore selection bias exists in that regard.Understanding the acquisition of gross motor milestones in the CDKL5 disorder is valuable information for caregivers, their therapists and clinicians and shows that for most, skills are attained at a much later age. It would be of interest to further investigate the events surrounding the acquisition of these milestones, especially with regard to seizure control. For caregivers, our data provide hope that their young child may still attain milestones at a later age than is usual for the general population. As the database grows, it will be important, to further examine the relationship between genotype and functional abilities. Individuals with the CDKL5 disorder have previously been described as having severely impaired development. Our findings show the variability within this disorder and suggest that there may be children with the CDKL5 disorder who present differently to the clinical picture originally described. Although we found no clear relationship between genotype and phenotype, the differences between individuals suggest that with greater numbers such a relationship may be identified. Continued research into the natural history of the CDKL5 disorder is needed to further our understanding of the variability seen within this condition.