Although migrant workers account for the majority of newborns in Beijing, their children are less likely to undergo appropriate universal newborn hearing screening/rescreening (UNHS) than newborns of local non-migrant residents. We hypothesised that this was at least in part due to the inadequacy of the UNHS protocol currently employed for newborn babies, and therefore aimed to modify the protocol to specifically reflect the needs of the migrant population.A total of 10,983 healthy babies born to migrant mothers between January 2007 and December 2009 at a Beijing public hospital were investigated for hearing abnormalities according to a modified UNHS protocol. This incorporated two additional/optional otoacoustic emissions (OAE) tests at 24–48 hours and 2 months after birth. Infants not passing a screening test were referred to the next test, until any hearing loss was confirmed by the auditory brainstem response (ABR) test.A total of 98.91% (10983/11104) of all newborn children underwent the initial OAE test, of which 27.22% (2990/10983) failed the test. 1712 of the failed babies underwent the second inpatient OAE test, with739 failing again; thus significantly decreasing the overall positive rate for abnormal hearing from 27.22% to 18.36% ([2990–973 /10983)]; p = 0). Overall, 1147(56.87%) babies underwent the outpatient OAE test again after1-month, of whom 228 failed and were referred for the second outpatient OAE test (i.e. 2.08% (228/10983) referral rate at 1month of age). 141 of these infants underwent the referral test, of whom 103 (73.05%) tested positive again and were referred for a final ABR test for hearing loss . Only 54 infants attended the ABR test and 35  were diagnosed with abnormal hearing.Our study shows that it is feasible and practical to achieve high coverage rates for screening hearing loss and decrease the referral rates in newborn babies of migrant workers, using a modification of the currently employed UNHS protocol. Hearing loss is a major sensory deficiency, which affects audiological development and impairs the quality of life of those affected ,2. One iUniversal newborn hearing screening (UNHS) is an effective way of identifying hearing loss in newborns , and in Despite the relatively high socioeconomic impact of the internal migrant sub-population in China, to our knowledge no study has reported the screening outcomes including the drop-out rates among this sub-population, using the recommended UNHS protocol for China. We have presumed that although the currently recommended UNHS protocol is suitable for the urban resident population, it is not optimal for internal migrants for many of the reasons stated above. The aim of our study was thus to design a specific UNHS protocol based on the local social characteristics of internal migrants, such that it would improve the screening rates for hearing loss in their newborn babies. This protocol would additionally allow detection of infants suspected to have permanent childhood hearing impairment (PCHI), in order that appropriate intervention and rehabilitation can be provided at an early stage.A total of 10983 babies , born at Beijing’s Shangdi Hospital between January 2007 and December 2009, were investigated. All babies were from migrant families (parents neither have registered permanent residence nor stable work in Beijing) and appeared to be normal and healthy at birth.Testing for hearing impairment was performed according to a modification of the recommended UNHS procedure, which incorporated an additional inpatient OAE test 24–48 hours after birth and an additional outpatient OAE test at 2 months after birth  test 24–48 hours after birth. When a baby tested positive and could stay in the hospital beyond 48 hours, it was referred to a second OAE test 48–72 hours after birth (this is the inpatient screening time in the recommend protocol). When a baby tested positive at the first test and did not undergo the second test, or when a baby tested positive at both tests as an inpatient, it was referred for an outpatient OAE test, at 1 month after birth. All infants testing positive after 1month were referred to an additional OAE test at 2 months after birth. Irrespective of whether or not an infant had undergone the OAE test after 2 months, all infants testing positive after 1 month were referred to a specialist hospital for an auditory brainstem response (ABR) test, for confirmation of a diagnosis of hearing impairment. When an infant was identified as having abnormal ABR, a further series of audiology tests; including OAE, ABR (AC and BC), 1 kHz tympanometry and CT (if necessary); were performed before 6 months of age, and appropriate intervention and rehabilitation were suggested to his/her parents/guardians.One of six qualified audiologists performed the first screening 24 to 48 hours after birth using the Transient Evoked Otoacoustic Emissions (TEOAE) test in the well-baby nursery. A Capella OAE equipment  presenting nonlinear clicks at 75dBpeak equivalent sound pressure level (SPL) was employed for this test. The TEOAE test was post-windowed by a 3 ms windowing function to suppress muscular and respiratory artefacts. Each recording was the mean result of 2080 sweeps; with minimal pass criteria being 50% reproducibility, 10dB SPL at emission response, and 3dB signal-to-noise ratio at any 3 analysis frequencies . All OAE recordings were performed in a sound shielded room during the baby’s natural sleep, after the baby had been fed.A diagnostic assessment for impaired hearing was finally performed by auditory brainstem response (ABR) testing, using an ICS Charter Evoked Potential equipment  in an electrical- and sound-shielded room. The settings employed for the ABR test were as shown in Table All procedures were performed with the consent of the parent/guardian. The study protocol was approved by the Ethics Committee of Beijing Institute of Otolaryngology and performed in accordance with the guidelines of the World Medical Association’s Declaration of Helsinki.p values < 0.05 were considered to be statistically significant.Statistical analysis was performed using the SPSS software . Chi-square test was used to detect significant differences in pass/refer rate between different steps of UNHS, and two-sided 2=244.906, p = 0))  of newborn babies were tested for hearing impairment at the first inpatient screening, ≤48 hours after birth. Overall, 72.78% (7993/10983) of the babies passed newborn hearing screening, whereas 27.22% (2990/10983) of the babies were referred for a second inpatient hearing test 48–72 hours after birth. The parents/guardians of 57.25% (1712/2990) of these babies agreed to the second inpatient OAE test, following which 56.83% (973/1712) passed; thus significantly decreasing the overall positive rate from 27.22% (2990/10983) to 18.36% ([2990–973 /10983)]; χ/11104 ofwere thus referred to a final confirmatory click ABR hearing impairment test  vs. referral rate of 2.08% at 1month of age)  infants suspected to have a hearing impairment 48–72 hours after birth, only 1147 (56.87%) underwent the OAE hearing test again after1-month at the hospital of their birth. Of these, 919 (80.12%) infants passed the test whereas 228 (19.88%) tested positive again and were referred for an additional OAE test at the same hospital at 2 months of age  at 1month of age). 141 of these 228 infants accepted the referral, of whom103 (73.05%) tested positive again by OAE test and  infants Overall, of the 190 infants referred, parents and guardians of 84 agreed for their children to undergo the final ABR test at 2 months after birth. Only 54 of these infants attended the test and 35 were confirmed positive for impaired hearing. Of these 35 infants SNHL, 12 had bilateral hearing loss ranging from mild to profound and 23 had unilateral hearing loss .Many countries have adopted early hearing detection programs over the past 30 years; with several UNHS protocols such as single OAE and ABR screening, OAE followed by ABR, or AABR (Auto ABR) with a follow-up ABR, etc. being employed . In ChinIn the current investigation we modified this UNHS protocol to better address the needs of newborn babies of internal migrants, who have steadily increased in numbers over the last decade in Beijing and often miss out on this important screening service due to the geographic instability resulting from their parents’ way of life. In particular postpartum, families are often unable to accept regular hospital appointments due to their frequent moves, and are reluctant to commit themselves to rescreening tests at the same hospital where the baby was delivered, despite availability of free screening/rescreening tests. Indeed, a relatively lower rescreening rate of 56.87% in the current study was attributable to three major factors; namely i) some babies could not be rescreened because they had been sent back to their parent’s home provinces after a few days, ii) some babies were rescreened in hospitals closer to the their new rented accommodation and relevant data were not available to the investigators at Shangdi Hospital, and iii) some parents were reluctant to accept re-testing because their new rental location was far away. These findings thus emphasize the importance of revising currently recommended hearing screening protocols for newborn babies of frequently migrating parents. Based on previous research , our revUsing this modified protocol, we have demonstrated that there was increased coverage rate in the target population, the basic measure of screening efficiency. In a hospital such as Shangdi Hospital, where the majority of pregnant women are poor migrants without any form of health insurance and therefore eager to leave as soon as possible to avoid the expense of hospital stay, we shortened the inpatient screening time from the normal 72 hours to a maximum 48 hours by adding an earlier OAE screening test at 24-48 hours to ensure higher coverage/acceptance rates for hearing screening. Indeed, on reviewing our database we found that 2169 infants would have missed the inpatient hearing screening based on the recommended protocol. Moreover, as 492 of these infants did not pass the inpatient hearing screening at 24–48 hours, under the recommended protocol these infants with suspected hearing impairment would have been missed if they had left the hospital by about 48 hours. Using this modified protocol, the coverage rate was found to be high at 98.91%, meeting the recommended coverage by the Joint Committee of Infant Hearing (JCIH) , and wasIn the modified UNHS protocol we kept the recommended inpatient OAE test at 48-72hours for referred infants staying in the hospital beyond 48 hours, to decrease the false positive rates. Although the present study indicated that the positive rate decreased significantly from 27.22% to 18.36% after the second inpatient OAE test 48–72 hours after birth, this was still much higher than the rate of 6.39% observed at the obstetric hospital downtown . This diThe referral rate is another important measure of a screening program’s efficiency and effectiveness and, according to the JCIH 2007 position paper, can be minimised by effective rescreening . Indeed,Of the 10983 migrant newborn babies that had the first hearing screening test, only 0.32% (35/10983) was found to have SNHL. The proportion of infants with identified unilateral n = 23) to bilateral (n = 12) hearing loss in our study was similar to that previously reported in developing countries; approximately 1:3–4  to bilat,29. HoweAlthough rescreening tests are the most important method for decreasing the false positive and referral rates, low rescreening rates have generally been a problem in newborn hearing screening programs ,28,29, aThe authors report no conflicts of interest.Further modifications and better design of the UNHS protocol reflecting socioeconomic factors specifically advantageous to particular populations may lead to significantly improved referral and rescreening.BQ collected and analyzed the data and contributed to writing the paper. XC collected, analyzed the data. HE collected the data. BL designed the study. SP collected the data. YZ collected the data. ZC collected the data. LH designed the study and analyzed the data. LZ designed the study, analyzed the data and contributed to writing the paper. DH designed the study. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2431/13/116/prepub


Numerous changes continue to occur in cochlear implant candidacy. In general, these have been accompanied by concomitant and satisfactory changes in surgical techniques. Together, this has advanced the utility and safety of cochlear implantation. Most devices are now approved for use in patients with severe to profound unilateral hearing loss rather then the prior requirement of a bilateral profound loss. Furthermore, studies have begun utilizing short electrode arrays for shallow insertion in patients with considerable low-frequency residual hearing. This technique will allow the recipient to continue to use acoustically amplified hearing for the low frequencies simultaneously with a cochlear implant for the high frequencies. The advances in design of, and indications for, cochlear implants have been matched by improvements in surgical techniques and decrease in complications. The resulting improvements in safety and efficacy have further encouraged the use of these devices. This paper will review the new concepts in the candidacy of cochlear implant. Medline data base was used to search articles dealing with the following topics: cochlear implant in younger children, cochlear implant and hearing preservation, cochlear implant for unilateral deafness and tinnitus, genetic hearing loss and cochlear implant, bilateral cochlear implant, neuropathy and cochlear implant and neural plasticity, and the selection of patients for cochlear implant. The first pediatric cochlear implant program was established at the House Ear Institute in 1980. Incredible as it may seem from the current perspective, the primary issue in that era was whether to consider implanting children at all. In 1980, the first child (a 9-year-old boy) was implanted, and by 1982, 12 children with age ranges from 3.5 to 17 years had been implanted in their program [Numerous clinical trials have been conducted by the FDA since cochlear implants were first introduced, and numerous supplements have been submitted to the FDA as these devices have undergone technological improvements. Thus, the determination of the implant candidacy is ultimately based on the best knowledge and judgment of the managing physician. Changes in candidacy have primarily included implanting persons with increasing amounts of residual hearing, implanting persons with increasing amounts of preoperative open-set speech skills, implanting children at younger ages, and implanting greater numbers of persons with abnormal cochleae. The primary reason that selection criteria have changed is that patients with implants are obtaining increasing amounts of open-set word recognition with the available devises. Although this increased performance is largely due to the technological advancements that have occurred in the field, it may also due to, at least in part, to the fact that patients with greater amounts of residual hearing and greater amounts of residual word recognition skills are receiving cochlear implants. The candidacy requirements for pediatric, and adult, cochlear implantation have gradually loosened. The obvious goal is to never have a single patient perform more poorly with their cochlear implant than they previously performed with hearing aids alone –4. Thus,Widespread universal newborn hearing screening has led to increased identification of infants with hearing loss worldwide –7. This For many years, the lower limit for age at implantation was 2 years. The first clinical trial to evaluate a multichannel implant in children was the Nucleus 22 device. The candidacy guidelines for this trial indicated appropriate candidates could be as young as 2 years of age. The first clinical trial to include children less than 2 years of age was with the Clarion device. The Clarion device could be indicated for children of 18 months if the Rx demonstrated ossification. All cochlear implant devices can be safely indicated for children of 12 months and older. As of 2000, the FDA has approved a device for implantation for those patients aged 12 months and older . AlthougFurther reductions in age at implantation are currently limited by the nature of audiologic testing in very young children. In general, using modern techniques, a confident assessment of severe to profound sensorineural hearing loss can be made in a child by the time they are 12 months old. In cases of hereditary hearing loss or meningitis, a confident assessment can sometimes be made at ages younger than this. Some authors , 6 prefeInherent in the discussion of CI in children younger than 12 months is the ability to reliably diagnose children of this age group with bilateral profound deafness. With respect to diagnosis of infant hearing loss, a paramount concern is the issue of specificity: the risk of implanting a child without profound deafness. CI evaluation of a child younger than 1 year of age should include the following: an attempt at behavioral audiometry , bilateral OAEs, ear-specific and frequency-specific ABR or ASSR, bilateral tympanometry, and acoustic reflexes. Present OAEs with abnormal ABR or ASSR and absent reflexes should generate suspicion for AN/AD, and ECoG can be used as an additional test in this setting –7.Speech perception testing in this age group remains challenging as most tests are language based and not appropriate for children younger than 1 year. use of the infant-toddler meaningful auditory integration scale (IT-MAIS), a parental survey of early speech development, has been employed reliably in this age group as a proxy for speech perception and linguistic development , 8. ThisAlthough used in a variety of research and settings, babble assessment is not currently a standard component of preoperative CI evaluation in children younger than 1 . On the Although anesthetic concerns unique to very young children exist, data in the CI literature support perioperative safety in the under-1 population. Lesinski-Schiedat et al.  reportedEven though the anatomical aspects of the facial recess and the cochlea are similar to older children, several risks are reported in the literature for cochlear implant in the younger group. Intraoperative blood loss, facial nerve anatomy, receiver/stimulator fixation, and device migration with skull growth present unique risks to children younger than 1 year . Two souLong accepted as a standard in temporal bone surgery, the use of facial nerve monitoring in CI surgery is nearly universal. In children younger than 1 year, minimal inferior extension of the postauricular skin incision, careful identification of the facial nerve, and judicious use of intraoperative facial nerve monitoring can assist with these surgical challenges. In very young children, the posterior scalp flap is typically thinner and more delicate, necessitating constant care and atraumatic handling of the skin flap and soft tissues . A varieThe circumferential dural exposure for seating of the receiver/stimulator is preferable for some authers so as to minimize device profile and damage from external trauma . Others Ongoing advances in receiver/stimulator design, such as the recently introduced Nucleus 5 (CI512) series by Cochlear, attempt to address these issues by creating a thinner device contoured to the infant skull. The effect of this change in device design is as yet unknown . Skull gPrograming the cochlear implant in young children is another challenger. Optimal use of CI technology requires an accurate assessment of threshold and comfort levels, obtained in older children and adults with behavioral testing. As with diagnostic testing, behavioral methods are frequently inappropriate for the very young child . Neural In very young children, ECAP measurements obtained through NRT/NRI/ART have been used successfully as a basis for threshold (T) and comfort. In very young children, ECAP measurements obtained through NRT/NRI/ART have been used successfully as a basis for threshold (T) and comfort (C) levels in creation of initial speech processor MAPs , 19.Many authors have provided evidence for long-term safety in the under-1 population. Roland et al. , 2009 reAt this point it is crucial to bring for discussion that CI in very young children is a safe and effective procedure. There is growing body of literature supporting improved auditory and linguistic outcomes in children implanted before 12 months of age , 20, 21.In conclusion, CI in the very young child provides unique challenges in diagnosis and certainty of testing, anesthetic risk, surgical technique, intraoperative testing and postoperative programming, long-term safety, development of receptive and expressive language, and speech perception outcomes. Overall, research to date support-minimal anesthetic, surgical, and long-term complications, suggesting that early implantation poses minimal risk to children younger than 1 year of age. Benefit in areas of receptive and expressive language development and speech perception has been suggested by multiple studies.This section will concentrate on research done in attempt to summarize strategies and results around the world in hearing preservation during cochlear implantation. This attempt has significantly changed the candidacy criteria in the last years . A numbeThe approach involves preserving existing residual acoustic hearing (low-frequency) in an ear to be implanted with a cochlear implant and then adding electrical stimulation via this cochlear implant for the missing high frequencies to produce speech understanding (and other hearing sensations) via combined acoustic and electric hearing (A + E).Word recognition research in patients with varying degrees of hearing loss by Turner et al.  among otMore recently, however, preservation of residual acoustic hearing in the implanted ear has received attention. Preservation of hearing within the implanted ear has been reported in several animal studies from the 1990s. Ni et al. . in 1992Hodges et al.  in 1997 Some groups have attempted to preserve residual acoustic hearing using a standard-length electrode that is partially inserted into the cochlea, combined with “soft surgery” techniques designed to minimize trauma. Gstoettner et al. in 2004  describePoor-frequency resolution and pitch perception of the traditional cochlear implant can lead to deficits in music perception. While implant listeners generally are quite good at perceiving the rhythmic cues in music , 27, theGstoettner et al.  tested nThe preservation of residual hearing has been shown to be practical and effective solution for severe high-frequency hearing loss . It can Up to now, treatment modalities of unilateral deafness consist of no treatment, conventional contralateral routing of signal (CROS), or bone-anchored hearing aid (BAHA) hearing aid. Cochlear implantation makes a new treatment modality available for patients with single-sided deafness. Arndt et al.  have recThe authors found that cochlear implantation improved hearing abilities in people with single-sided deafness and is superior to the alternative treatment options. The use of the CI did not interfere with speech understanding in the normal-hearing ear. Their data suggested that the binaural integration of electric and acoustic stimulation is possible even with unilateral normal hearing.Tinnitus is a frequent and often debilitating condition, which is difficult to treat. The most frequently used therapies consist of auditory stimulation and cognitive behavioural treatment aiming at improving habituation and coping strategies. However, more causally oriented therapeutic strategies are lacking and need to be developed to relieve auditory perception disturbances. Even though the pathophysiology of tinnitus remains incompletely understood there is increasing evidence that tinnitus is related to alterations of neuronal functioning in the central auditory system. Like phantom limb pain, tinnitus as an auditory phantom perception seems to be the correlate of maladaptive attempts of the brain at reorganization due to distorted sensory input. This notion is confirmed by the finding that hearing loss is the most important risk factor for developing tinnitus and that most people with sudden unilateral deafness experience tinnitus. Animal experiments have demonstrated that reduced auditory input causes a dysbalance between inhibitory and facilitatory mechanisms throughout the central auditory pathways, which then results in reorganization of the tonotopic maps in the auditory cortex. This might represent a neuronal correlate of tinnitus . AccordiIn subjects who are deaf and who also have tinnitus in the affected ear, tinnitus treatments based on acoustic input are impossible. On the other hand, tinnitus suppression using electric stimulation has been reported to be successful. Buechner et al.  have iniIn 3 participants, the tinnitus was significantly suppressed while wearing the device. In the other 2 participants, the tinnitus could be reduced in certain situations. Speech perception tests revealed a significant benefit with the CI in combination with the normal-hearing side for 3 participants. All participants accepted the device in a clinical setting; adaptation of the frequency allocation was not required. They concluded that improvements were found regarding the hearing and the tinnitus. Not all participants benefit from the CI to the same degree and in the same situations. The results indicate that cochlear implantation in subjects with unilateral severe to profound hearing loss and ipsilateral tinnitus may be beneficial on a case-to-case basis. However, further work needs to be performed to define the appropriate indication criteria for selection of patients with unilateral deafness.Kleinjung and colleagues  reportedThe application of cochlear implants for tinnitus relief in patients with unilateral deafness has so far been described in another study . All 21 According to different pathologic changes that generate neural activity interpreted as tinnitus, there are several possible mechanisms which may account for tinnitus suppression after cochlear implantation. Some reports further support the model of tinnitus pathophysiology, in which chronic tinnitus as an auditory phantom perception might be the correlate of maladaptive attempts at cortical reorganization due to peripheral deafferentation . As a coResidual inhibition might explain tinnitus suppression effects which outlast the active stimulation period for a certain amount of time. But some observation that tinnitus was neither perceived in quiet environments nor during sleep might not entirely account for this theory. The effect of the insertion of the electrode into the cochlear should also be discussed . CochleaIn summary, disabling tinnitus resulting from sudden unilateral deafness should be considered as a new indication for a cochlear implant procedure. As demonstrated in this report and supported by the literature data, cochlear implantation may represent a chance for complete suppression of tinnitus in selected patients.The hereditary causes are responsible by greater than 60% of all prelingually deafened with environmental or iatrogenic causes responsible by the remaining 40% . Some ofConnexin 26 (Cx26) mutation has been reported to cause 50% of cases of nonsyndromic autosomal recessive hearing loss which makes this disorder the most common cause of nonsyndromic hereditary hearing loss. Dominant Cx26 mutations have been associated with syndromic disease with skin disorders such as keratitis ichthyosis deafness syndrome and palmoplantar keratoderma with deafness, and they are rare . The connexins are proteins. Six containes assemble to form half a channel or connexon and two connexons together create an intracellular gap junction channel involved in electrolyte transportation keeping the potassium gradient within the spiral ligament and stria vascularis and communication between cells too. When these proteins present a defect may, occur potassium accumulation, and lack of recirculation is presumed to lead to hair cell dysfunction and degeneration . AnotherUsher syndrome is one of the most common cause of deaf blindness in humans , and it The literature describes three types of the Usher syndrome. The type I (USH1) is the most severe form of the Usher syndrome and well discussed, accounting for 30–40% and it is characterized by severe to profound congenital hearing loss, motor development delay in children, and progressive retinopathy with vision loss, decreased peripheral vision, and central acuity in the first decade of life leading to blindness by young adulthood. Molecular studies have shown that the defective proteins are located within the developing auditory hair bundle, either within the stereocilia or kinocilium. The defective proteins in USH1—myosin VIIa, PDZ-domaincontaining protein harmonin, cadherin 23, protocadherin 15, and the scaffolding protein Sans—are hypothesized to be associated with hair bundle-linked-mediated adhesion forces . In temporal bone examination from USH1 patients, severe degeneration primarily of the basal turn of the organ of Corti, atrophy of the stria vascularis, and a decrease in spiral ganglion cells were found. The cochlear neurons were diminished with an average of 68% neuronal loss compared with age-matched controls . Patient+ K+ ATP pumps. It is hypothesized that mitochondrial dysfunction results in ionic imbalances, cell injury, and then death with concomitant hearing loss. The basal aspect of the cochlea, which is responsible for high-frequency hearing, requires even greater metabolic support. As a result, early injury to this area like in aminoglycoside ototoxicity or noise exposure results in the classical high-frequency hearing loss associated with mitochondrial dysfunction, which slowly progresses to affect other areas of the cochlea [Mitochondrial DNA (MtDNA) mutations are maternally inherited predominantly, because mitochondria are located at the cytoplasm, and the fertilized embryo receives almost only the ovum cytoplasm. The mitochondria is involved in adenosine triphosphate (ATP) production through oxidative phosphorylation process that is critical especially for those organs with high metabolic needs. At the inner ear outer hair cells and the stria vascularis have high ATP demands. The hair cells rely on an appropriate endocochlear potential produced by the stria vascularis and its many Na cochlea , 51, and cochlea , 53. Since sensorineural hearing loss (SNHL) is found in 42% to 70% of patients with mitochondrial disorders and cochlear implants are the treatment choice for a large number of them, it becomes relevant to look at this disease group. In these patients hearing loss is classified as syndromic: mitochondrial encephalopathy, lactic acidosis and stroke-like episodes (MELAS syndrome), maternally inherited diabetes and deafness (MIDD syndrome), the Kearns-Sayre syndrome and chronic progressive external ophthalmoplegia (CPEO) and nonsyndromic. The nonsyndromic SNHL is associated with ototoxic side effect of aminoglycoside antibiotics and the predominant mutation at maternally inherited MtDNA is A1555G in more than 50% of patients that present SNHL after aminoglycoside use. SNHL occurring in patients with the A1555G mutation without aminoglycosides exposure is reported, but it is milder. Residual hearing is found in patients with A1555G mutation associated with lower thresholds for electric promontory stimulation that suggests spiral ganglion cell preservation, making them good candidates for cochlear implants. Usami et al.  related The A3243G mtDNA mutation, as well as more common large-scale deletions, has been found responsible in skeletal muscle biopsy specimens, and these are usually of sporadic origin in both KSS and CPEO , and botWaardenburg syndrome (WS) is an autosomal syndrome characterized by dystopia canthorum, hyperplasia of the eyebrows, heterochromia iridis, a white forelock, and variable sensorineural hearing loss present in 1 in 40,000 live births and responsible by 2–5% of all congenitally deafened children . Four feAbsence of melanocytes affects pigmentation of the hair, skin, and eyes as well as the neural crest cells that migrate and form the basis of the stria vascularis , 61. HeaKCNQ1 and KCNE1 (LQT1) genes that form the slow component of the delayed rectifier potassium channel complex . The Jervel Lange-Nielsen syndrome (JLNS) is characterized by a constellation of syncope, sudden death, congenital sensorineural deafness, and cardiac arrhythmias like significant bradycardia, ventricular tachycardia, ventricular fibrillation, and torsades des points, which leads to syncope and sudden death if not treated . 95% of This mechanism is responsible by endolymph potassium maintenance at the stria vascularis and ventricular repolarization by moving potassium ions out of the cell , 67. ScaOur auditory environments are noisy and full of multiple sound sources that are a challenge for auditory system. The binaural system is responsible for providing cues that segregate target signals from competing sounds, and it is able to identify sound sources in normal hearing listeners. Binaural hearing is the result of integration between inputs from the two ears and auditory pathways. The consequence of binaural hearing is the speech understanding when competing sounds are present, and it is well known that when listening with only one ear, sound localization becomes very difficult to achieve. Three primary effects on perception have been identified in binaural hearing: the head shadow effect, the binaural summation effect, and the binaural squelch effect . The head shadow effect occurs during everyday listening conditions when speech and noise are spatially separated. For example, background noise coming from the right side would interfere with the right ear but the head would block (create an acoustic shadow) some of the interfering noise from reaching the left ear. Thus, the head shadow effect would result in a better signal-to-noise ratio (SNR) in the protected left ear . A listeThe head shadow effect has the largest impact on hearing with binaural cochlear implantation. The binaural squelch effect also operates when competing noise is spatially separate from the signal so that the two ears receive different inputs. Unlike the purely physical head shadow, however, squelch requires central auditory processing that integrates the signals from each ear so that the auditory cortex receives a better signal than could be possible from either ear alone . The braBinaural summation also refers to a central processing effect, but it is thought to occur when both ears are presented with a similar signal. The combined signals from the two ears are perceived as louder by up to 3dB compared with monaural listening to the same signal . This doConsidering the advent of cochlear implantation and the great benefits experienced by users, it is important to stress its limitations, some of them from the device itself and other ones associated to monoaural hearing. The unilateral cochlear implant user shows poor ability in sound source identification, and it could be difficult hearing speech in noisy environment, so if the bilateral cochlear implant recipients are able to utilize the effects described above, this ultimately determines the degree of additional benefit a second implant will provide. The literature now containes huge series of bilateral cochlear implantation, and authors reported the ability of bilateral CI users to hear speech in the presence of competing stimuli and they have advantage of spatial separation between target and competing speech –77. A coLocalization and speech-in-noise abilities in this group of patients are somewhat correlated . In bilaThe specialists were concerned about unilateral cochlear implanted difficulties in everyday listening conditions. Functional localization of sounds is not possible with only 1 implant, often creating a safety issue, and hearing in noise is very difficult. They consider bilateral cochlear implantation advisable based on the results of improved speech intelligibility and sound localization and the expansion of the receptive sound field. Finally they agree that both children and adult may have better auditory performance with bilateral implants compared with unilateral ones . Bilateral cochlear implants may not be an option or recommended for all adult recipients. This could be due to health issues that prevent a second surgery, lack of insurance coverage, or, in many cases, a notable amount of residual hearing in the nonimplanted ear. In these cases, the use of a hearing aid in the nonimplanted ear can represent a viable, affordable, and potentially beneficial option for bilateral stimulation despite the asymmetry in hearing between ears. This asymmetry occurs because the type of auditory input received by the two ears is quite different. The cochlear implant provides electric stimulation, while the hearing aid provides acoustic stimulation, which in combination across the ears is likely to provide atypical interaural difference cues in time and level . PerformWhen considering the best moment to do the second implant, surgeons should inform patients about the same risks from the first operation and that the additional benefits with the second one may be not so great (it only increases around 20%) . This waGordon et al.  have exaAuditory neuropathy/dyssynchrony (AN/AD) is a form of hearing impairment characterized by moderate-to-profound sensorineural hearing loss, progressive or transient, in which function outer cells is preserved, but afferent neural activity in the auditory nerve and central auditory pathways is disordered . The incThe auditory pathway disorder is suggested by the absence or severe distortion of electric potentials from the auditory nerve  and auditory brainstem response (ABR) . There aThe incidence of AN has been estimated as 10% to 14% of children diagnosed with severe to profound SNHL . The natn = 80). Many of the children have proceeded to successful implantation, with a smaller number failing to gain significant benefit [The Sydney Cochlear Implant Centre (SCIC) has one of the largest cohorts of pediatric patients with AN undergoing cochlear implantation in the world   was condThe removal or inactivation of cochlear hair cells leads to an immediate loss of activity in the auditory nerve . The strPhysiologically, neurons that normally only receive excitation from a deafened ear become inactive following IHC inactivation at any age, but, paradoxically, those in other parts of the auditory system become hyperexcitable , a respoIt is well known that auditory perception of cochlear implant users gradually improves after post-surgical activation, and this improvement occurs with variable extent in different age groups, over different time courses, and for different outcome measures . The litIn recent years the indication criteria for cochlear implantation (CI) have changed. To gain optimal benefits, early implantation in prelingually deaf children is necessary. Even additional disabilities are no longer contraindications for CI. Nowadays the criteria for implantation not only include deafness but also residual hearing. Combined electric-acoustic stimulation has been established as a treatment option in patients with hearing still functioning in the low frequencies. Due to the benefits of binaural hearing, bilateral CI has become standard over the last decade. Recent experience has shown the benefits of CI in unilateral deafness and in cases of severe tinnitus. The actual benefit of CI shows great interindividual differences. We usually expect (re-)habilitation of language communication skills with implantation.


M value of up to 9 mM and seems to be activated by iron [M value in the range of 100-300 µM and is additionally degrading 2’,3’-cCMP as well as the 2’,3’- and 3’,5’-isomers of cUMP, cAMP, cGMP and cIMP. The multifunctional cCMP PDE is inhibited by inorganic phosphate and AMP. Both enzymes are calmodulin-insensitive, IBMX-resistant and show a low molecular weight of <35 kDa, which is uncommon for the “classic” phosphodiesterases. However, the exact amino acid sequence and identity of these proteins remain elusive.The literature of the past four decades contains several scattered reports that demonstrate the existence of two different 3’,5’-cCMP degrading enzymes with distinct substrate affinity and specificity. The so-called “cCMP-specific” PDE was isolated from disrupted L-1210 leukemia cells [ by iron ,7 and hu by iron . This enOur project aims at a clear and unequivocal determination of amino acid sequence and identity of these elusive cCMP-degrading enzymes by analyzing and purifying the cCMP degrading activity of organ extracts and biological fluids (e.g. serum). Moreover, we re-investigate well-known “classic” PDEs in enzymatic assays to identify a potential 3’,5’-cCMP degrading activity.The substrates and products of PDE reactions are detected and quantified with high accuracy by a highly sensitive HPLC-MS/MS method (triple quadrupol mass spectrometer) which is established in our institute. This method enables us for the first time to unequivocally identify cyclic nucleotides and their degradation products in biological samples.We assay organ extracts and biological fluids for cCMP PDE activity and use the standard methods of protein biochemistry to identify, concentrate and purify these enzymes . A very important part of our methodology is the use of an FPLC system for protein separation. The identity of purified proteins will be determined by analyzing a fingerprint of the fragments in mass spectrometry and comparing the results with the content of biological databases. In addition, we characterize the enzymatic activity of commercially available recombinant PDEs.A screen of 13 commercially available recombinant PDEs revealed that these enzymes show unexpectedly broad substrate specificity and also degrade “exotic” cyclic nucleotides like cUMP, cIMP, cXMP and cTMP . Most inIn addition, we have identified a heat-sensitive cCMP-degrading activity in certain commercially available batches of fetal bovine serum. This enzymatic activity can be considerably enhanced by filtration of the serum through centrifugation filtration devices (30 kDa cutoff). Results from FPLC chromatography suggest that this enzyme may be associated with larger protein complexes, one of the components probably being serum albumine.Our PDE 7A1 data and the heat-sensitive cCMP-degrading activity detected in bovine serum samples demonstrate that cCMP PDEs really exist. We plan to characterize PDE 7A in more detail in enzyme kinetics experiments using fluorescent cNMP analogues. Moreover, we plan to purify and concentrate the cCMP-degrading activity in bovine serum in order to further characterize it with various substrates and inhibitors. In addition, we will also test organ extracts, specifically from liver (mouse and pig) in order to confirm the results previously reported in the literature and to finally reveal the identity of the “cCMP-specific” and “multifunctional” cCMP-PDE.


Potential methods of application of self-administered Web-based pure-tone audiometry conducted at home on a PC with a sound card and ordinary headphones depend on the value of measurement error in such tests.The aim of this research was to determine the measurement error of the hearing threshold determined in the way described above and to identify and analyze factors influencing its value.The evaluation of the hearing threshold was made in three series: (1) tests on a clinical audiometer, (2) self-tests done on a specially calibrated computer under the supervision of an audiologist, and (3) self-tests conducted at home. The research was carried out on the group of 51 participants selected from patients of an audiology outpatient clinic. From the group of 51 patients examined in the first two series, the third series was self-administered at home by 37 subjects (73%).The average difference between the value of the hearing threshold determined in series 1 and in series 2 was -1.54dB with standard deviation of 7.88dB and a Pearson correlation coefficient of .90. Between the first and third series, these values were -1.35dB±10.66dB and .84, respectively. In series 3, the standard deviation was most influenced by the error connected with the procedure of hearing threshold identification (6.64dB), calibration error (6.19dB), and additionally at the frequency of 250Hz by frequency nonlinearity error (7.28dB).The obtained results confirm the possibility of applying Web-based pure-tone audiometry in screening tests. In the future, modifications of the method leading to the decrease in measurement error can broaden the scope of Web-based pure-tone audiometry application. The development of Internet technologies combined with technological progress in the construction of personal computers, especially in terms of improving performance of sound cards, offer the possibility of conducting self-administered hearing tests at home. Moreover, in a group of elderly people who often suffer from hearing loss, there is an increasing number of Internet users. The validity of such hearing tests needs to be evaluated before applying the test results in the diagnostic and therapeutic process.The remote examinations of hearing conducted with the use of PCs have been carried out for about 10 years -4 and exThe hearing screening tests done remotely with the use of a PC sound card are most often in the form of a speech-in-noise test ,7,11,12.The potential application of pure-tone audiometry conducted on a PC depends on the value of the measurement error. The PC-based test will not substitute the clinical pure-tone audiometry. However, it can be applied for conducting self-administered check-ups in cases of limited access to clinical devices, eg, at the general practitioner. Alternatively, it can be used as an initial telemedical examination combined with a survey, which will help determine the direction of future treatment. The aim of the research was to identify the measurement error connected with determining the hearing threshold conducted by means of self-administered Web-based pure tone audiometry, as well as identify and analyze factors affecting its value.The hearing threshold evaluation was done in three series: (1) audiologist-performed tests on clinical audiometer at an audiology outpatient clinic, (2) self-administered Web-based tests on a specially calibrated computer at an audiology outpatient clinic under the supervision of an audiologist, and (3) self-administered Web-based tests conducted at home.In series 1 and 2, 51 participants , aged 11-60 years (the median age was 34) underwent examination. The research participants were selected from among the patients of an audiology outpatient clinic. The qualification criterion was the willingness to participate in the research, owning a PC, basic skills to operate it, and having an email account. 102 ears were examined from which 45 (44%) were ears without a hearing loss, ie, with the hearing threshold of 25dBHL and less, 17 (16.7%) were ears with hearing impairment below 40dBHL, 31 (30.4%) with hearing impairment above 40dBHL and below 70dBHL, and 9 (8.8%) were above 70dBHL. From the group of 51 patients examined in the first two series, series 3 was self-administered at home by 37 subjects (73%). The examinations in series 1 and 2 were conducted on the same day, and examinations from series 3 were conducted up to 196 days later (median of 9 days). In the case of failure to conduct test in series 3 in a time of 2-3 weeks, the patients were reminded by a phone call and then after about 1 month, they were reminded by an email.All tests from series 1 were made in an acoustic cabin with the use of clinical audiometer Interacoustic AD229e and headphones TDH-39. The calibration of the audiometer was conducted according to ISO 389-1:1998. The hearing threshold in pure-tone audiometry was determined with the use of the ascending method, according to ISO 8253-1:2010. The level of the tone was reduced in 10dB steps until no further response occurred, and then it was increased in 5dB steps until the subject responded. The threshold was defined as the lowest level at which responses occurred in at least half of the series of ascending trials with a minimum of two responses required at that level . The exaThe examinations in series 2 and 3 were conducted on personal computers following their calibration. Both the calibration and the examination were performed at system volume set at the maximum level. The calibration consisted of determining the reference sound level by a person with good hearing. During calibration, two sounds differing in intensity were presented bilaterally in turns for 1 second at a frequency of 1kHz. The difference in sound intensity was stable and equalled 5dB. The task of the reference person was to set the volume in a way that ensured that only the louder of the two sounds presented was audible. The volume was controlled by means of a slider with the step of 1dB. The reference person could listen to the sound for unlimited time, adjusting the volume using the slider with 1dB step any number of times, in order to finally confirm the selected level with a button. The mean intensity of the two presented sounds determined the hearing level of the reference person and was marked 0dBRP-HL. The reference values at frequencies differing from the calibration frequency, ie, 1kHz, were calculated using the model based on the A-weighting filter .The procedure of determining the hearing threshold during the test, both in series 2 and 3, differed from that applied during calibration. The task of the subject was to set the intensity of the presented pure tone modulated by sinusoidal envelope with a 2-second period at such a level that the sound was on the verge of audibility. Patients could listen to the sound for unlimited time and change the intensity of the presented sound signal by themselves using a slider with 5dB step any number of times, and then confirm the chosen level with a button. The hearing threshold was determined at frequencies as in series 1, ie, 250Hz, 500Hz, 1kHz, 2kHz, 4kHz, and 8kHz. For hearing thresholds below 0dBHL-RP, the value was assumed to be 0dBHL-RP. The time spent on calibrating and examinations was not recorded.All the examinations in series 2 were conducted on a notebook Dell Vostro 1320 with operational system Microsoft Windows 7 and headphones Technics RP-F290 placed in an acoustic cabin of an audiology outpatient clinic and connected to the Internet. The calibration was repeated six times by 3 people whose hearing threshold in pure-tone audiometry at the calibration frequency was 0dBHL. Each person performed calibration twice. The final value of the calibration coefficient was determined as the mean of all values obtained from single calibrations and was the same for all examinations in series 2. Examinations were supervised by an audiologist, whose task was to train a patient and detect hisher mistakes, eg, changing the sides of headphones, omission of the frequency, or accidental marking of a threshold that was significantly different from the actual one.Examinations in series 3 were conducted by the patients themselves on their own personal computers at home. Each examination was conducted on a different computer using different headphones. The test station was calibrated by other household members with no previous hearing problems. In that series, the hearing threshold of the reference person was not controlled. The trial participants were instructed to conduct the test at home in a quiet place, preferably in the evening or at night. Moreover, they were informed that the calibration should be performed by a person up to 35 years old, with no previous hearing problems.The hearing threshold determined in series 1 was compared to hearing thresholds from series 2 and 3. If there was no response at a given frequency, measurement was not taken into account for further calculations. The mean difference between thresholds in series, its standard deviation, Pearson’s correlation coefficient, and linear estimators  were calThe mean difference between thresholds in series, its standard deviation, Pearson’s correlation coefficient, and linear estimators of Deming’s regression were determined for the hearing loss below 40dBHL as well as greater than or equal to 40dBHL . The divP=.05, and the specificity was 0.96 with confidence intervals of  at P=.05. For series 3, the respective values were sensitivity 0.89  and specificity 0.89 .On the basis of the obtained results, the sensitivity and specificity of noise-induced hearing loss detection was calculated, according to the criteria proposed in  adopted This paper attempts to identify and assess the values of the sources of error in determining the hearing threshold of Web-based examinations. The literature data on standard deviation of the hearing threshold determined in test-retest examinations carried out in clinical settings are presented in X and Y is the sum of their variance  at the level of literature data σclin, the standard deviation of the measurement error in Web-based hearing tests for series 2 σ(ii) was calculated at 6.64dB based on the value σ(i) and the value σ(i)(ii), which is the standard deviation of the hearing threshold difference between the values determined in series 1 and 2. Similarly, for series 3, we obtain the standard deviation σ(iii) equal to 9.78dB , both for series 2 and for series 3.gain≈0dB).With the increase in intensity, increment of the standard deviation of measurement error in series 3 is slightly smaller than in series 2. In series 2, the standard deviation of the gain error equals zero since all tests are done on the same set. The lack of increase in standard deviation in series 3 above the values observed in series 2 indicates a negligible effect of the gain error on the measurement values . Taking into account the above, we obtain the calibration error σcal equal to 6.19dB , the calibration error , and additionally at the frequency of 250Hz, the frequency response nonlinearity error (250Hzσnonlin=7.28dB).In series 3, the standard deviation values were most influenced by the error connected with the procedure of determining the hearing threshold, the same as the error in series 2  may be attributed to the difficulty connected with self-assessment of the hearing threshold. Standard deviations between series 1 and 2 as well as between series 1 and 3 increase together with the increase in the hearing loss  is connected with the error of hearing threshold assessment σproc_cal and the standard deviation in the hearing threshold of the reference persons σpop at the calibration frequency, ie, 1kHz. The standard deviation of the hearing threshold determined by means of the ascending method among the population of young subjects without prior hearing problems, σpop_asc, can be estimated on the basis of literature data [pop_asc=5.6dB. Assuming the standard deviation of the ascending method σclin is at the level of 4.25dB, we obtain σpop at 3.65dB and then σproc_cal at 5.00dB , despite the possibility of setting the threshold with the accuracy of 1dB step, is characterized by a slightly higher error than in the ascending method σclin=4.25dB, whose step equals 5dB. Therefore, accuracy improvement of the Web-based examination can be achieved by modifying the method. An interesting solution is offered by, eg, the Békésy’s method, as it is characterized by lower standard deviation in the test-retest examination compared to the ascending method [The method of the hearing threshold evaluation used during calibration  of the headphones used in the examination and the frequency nonlinearity of the sound card. The biggest differences between the RETSPL values occur at low frequencies [The frequency nonlinearity error is the difference between the actual values of the reference sound level and the values determined by the model. The greatest value of quencies , also inTaking into account the significant value of the calibration error and frequency nonlinearity error, it seems interesting to conduct pure-tone audiometry examination on generally available appliances with known electro-acoustic parameters, eg, smartphones. On determining frequency characteristics of the selected smartphone model with bundled headphones, it seems possible to omit the calibration stage and thus obtain more precise examination results.Web-based pure-tone audiometry can be used without previous training in its conducting. However, it requires the knowledge of basic terms such as the hearing threshold or frequency. On the other hand, an attractive, intuitive and user-friendly interface can largely replace training. Prior to the tests in series 3, patients were instructed on how to perform the test and had performed similar tests before in series 2 under the supervision of an audiologist. The knowledge of the application could lead to reduction in the value of the measurement error.The obtained results of sensitivity and specificity confirm the possibility of applying Web-based pure-tone audiometry in screening tests. Moreover, Web-based pure-tone audiometry may be used for self-monitoring of hearing, especially if tests are to be conducted under the same conditions. If the same equipment is used, the relative error between subsequent examinations will be reduced by frequency nonlinearity error, and in the case of the same calibration coefficients, relative error will be reduced by the calibration error. Self-monitoring of hearing may become applicable in hearing disorders, such as fluctuating hearing loss, tinnitus, sudden hearing loss, otosclerosis, Ménière’s disease, as well as during treatment with ototoxic drugs. In the future, modifications of the method leading to the decrease in measurement error can broaden the scope of Web-based pure-tone audiometry application.


Borrelia burgdorferi alternates between ticks and mammals, requiring variable gene expression and protein production to adapt to these diverse niches. These adaptations include shifting among the major outer surface lipoproteins OspA, OspC, and VlsE at different stages of the infectious cycle. We hypothesize that these proteins carry out a basic but essential function, and that OspC and VlsE fulfil this requirement during early and persistent stages of mammalian infection respectively. Previous work by other investigators suggested that several B. burgdorferi lipoproteins, including OspA and VlsE, could substitute for OspC at the initial stage of mouse infection, when OspC is transiently but absolutely required. In this study, we assessed whether vlsE and ospA could restore infectivity to an ospC mutant, and found that neither gene product effectively compensated for the absence of OspC during early infection. In contrast, we determined that OspC production was required by B. burgdorferi throughout SCID mouse infection if the vlsE gene were absent. Together, these results indicate that OspC can substitute for VlsE when antigenic variation is unnecessary, but that these two abundant lipoproteins are optimized for their related but specific roles during early and persistent mammalian infection by B. burgdorferi. Borrelia burgdorferi cycles between mammalian and tick environments, each of which varies over time  lipoprotein genes allowed spirochaetes to persist for several weeks after inoculation into SCID mice . The complementing plasmid was lost during persistent infection, indicating that ospC expression was not required at later times. In immunodeficient SCID mice, which lack B and T cells, the ospC-containing shuttle vector was more stable, most likely because these mice do not produce antibodies against OspC that could lead to clearance of spirochaetes that retained the plasmid and continued to express ospC. Nevertheless, significant shuttle vector loss was detected, indicating that sustained OspC production was not necessary during persistent infection of SCID mice either. In the present study, we reasoned that if VlsE normally fulfils the role of OspC during persistent infection, spirochaetes lacking VlsE would be unable to tolerate the absence of OspC during persistent infection. A measure of this intolerance would be strong positive selection for shuttle vector maintenance during persistent infection with a B. burgdorferi mutant lacking both the endogenous vlsE and ospC genes, but complemented with a shuttle vector carrying the ospC gene. Consequently, we screened for retention of the ospC-containing shuttle vector at a time at which we had previously found significant loss of the same shuttle vector by ospC mutant spirochaetes that retained lp28-1 and, therefore, vlsE. We predicted that spirochaetes lacking VlsE would retain the ospC-containing shuttle vector, in order to continue producing OspC in the absence of VlsE. Consistent with this hypothesis, a previous study with lp28-1-deficient spirochaetes found that these spirochaetes maintained OspC production during persistent infection of an immunodeficient host , a double mutant lacking both vlsE and ospC (vlsE−ΔospC), and the same two strains containing the ospC shuttle vector pCpospC (vlsE−/pCpospC and one WT mouse infected with vlsE−ΔospC/pCpospC (pospC were seropositive (vlsE) is required for persistent infection of WT mice.Wild-type  and C3H-SCID mice were inoculated with 10 pCpospC A. Six we pCpospC . Isolate/pCpospC . Howeverpositive , indicatvlsE but containing ospC either at its normal location or on a shuttle vector  persisted in all tissues in all SCID mice inoculated, whereas those lacking both ospC and vlsE (vlsE−ΔospC) were not isolated from any SCID mouse tissues  were isolated from most SCID mice in which they were inoculated. This result differed from previous work and other experiments in this study, in which ospC was found to be an essential gene for normal infection of both immunocompetent and immunodeficient mice . This result supports our hypothesis that continued OspC production would be required by bacteria lacking VlsE, if these two outer surface proteins fulfil the same essential function during infection of the mammalian host.If sustained OspC production is required when VlsE production is not possible, spirochaetes lacking both spC gene   and pCpospA  and pFpospA (flaBpromoter-ospA) encode constitutive vlsE and ospA expression from the flaB promoter. Finally, shuttle vector pCpvlsE2 (Cosppromoter-vlsE2) has the ospC promoter and signal sequence fused to the VlsE coding sequence , to test if lipoprotein processing and membrane localization affects how well VlsE can fulfil the role of OspC (Table ospC mutant B. burgdorferi (ΔospC) to determine if OspA or VlsE, appropriately regulated or constitutively expressed, could fulfil the requirement for OspC in establishing mammalian infection. The shuttle vectors were also introduced into WT B. burgdorferi to investigate if inappropriate expression of these lipoproteins in the presence of native ospC expression would be deleterious for the spirochaetes. Finally, we verified that expression of the CpvlsE and CpospA genes on the shuttle vector paralleled that of the endogenous ospC gene by introducing these constructs into the high-passage strain B313  attenuated WT B. burgdorferi at a dose of 104 in both WT and SCID mice , we tested whether the converse were true, and VlsE could fulfil the role of OspC in establishing mammalian infection. We first used shuttle vectors carrying the VlsE or OspA open reading frames (ORFs) under the regulation of the  vectors  and B. I vectors . All WT CID mice . This fiospC promoter, the previous study of Xu et al. found that constitutive expression of these proteins in an ospC mutant background could restore infectivity to varying degrees in both WT and SCID mice , as was observed by Xu et al. (ospC and the ΔospC/pFpospA strains in SCID mice (3/10 vs 5/5 mice infected respectively)  . Howeverctively) , surpris of vlsE . This maospC mutant was attempted with genes located on shuttle vectors, which generally have slightly higher copy number than the endogenous genomic plasmids of B. burgdorferi  was a result of excess protein production due to higher copy number, we constructed a strain in which the ospC gene located on cp26 was replaced with the vlsE coding sequence, with the fusion between the ospC promoter at the start codon of the vlsE ORF . Appropriate expression of the vlsE gene from the ospC promoter was assessed by growing spirochaetes in vitro at pH 6.8, in which conditions the WT ospC gene is typically induced. Although not entirely conclusive, a lysate of ΔospC::CpvlsE  grown in these conditions appeared to contain more VlsE protein than was found in WT spirochaetes, as expected [ΔospC::CpvlsE (pH 6.8), B. burgdorferi (data not shown), confirming that the ospC promoter was induced in the conditions used. When tested for the ability to infect WT or SCID mice at an inoculum of ∼ 103 per mouse, the ΔospC::CpvlsE bacteria were non-infectious in both cases , even when using the same vlsE allele as Xu et al.Although this study does not support the idea that VlsE can also substitute for OspC, some data do suggest that other , et al.  partiallospA was unable to fully complement an ospC mutation. Although we found apparent complementation of the ospC mutation by ospA driven by the flaB promoter in SCID mice, the ability of ΔospC spirochaetes to infect SCID mice on occasion  site.It remains unclear why OspC can substitute for VlsE but not vice versa. OspC may perform a unique role early in infection, in addition to a common function that VlsE typically assumes as the spirochaetes persist in a mammal. Without invoking an additional unknown role, VlsE could be unable to take the place of OspC because the lipoprotein requirement during initial infection by et al., et al., B. burgdorferi against host defences, which defences those might be remains undefined. We have looked without success for differences between ospC mutant and WT spirochaetes with respect to phagocytosis by mouse and human neutrophils, phagocytosis by mouse macrophages, susceptibility to human or mouse complement, or susceptibility to mouse natural killer cells . Although these studies were not exhaustive, they emphasized the complexity of the interaction between host and bacterium and the inherent limitations of investigating that interaction either with isolated components or in vivo. If OspC and VlsE stabilize the bacteria in the host environment, large amounts of a particular lipoprotein may be an essential component of the recently described B. burgdorferi lipid rafts . This is in contrast to the complete lack of infectivity by ospC mutant spirochaetes that we found in earlier studies , so we do not think that the ΔospC mutant used in this study has undergone a compensatory mutation. Since the earlier tissue transfer studies suggest that a B. burgdorferi protein produced during persistent infection, when appropriately regulated and in the correct context, can successfully fulfil the ospC requirement   or NEB5α .Samuels, , using 5ospC gene in −vlsE, which lacks lp28-1 and the encoded vlsE locus  that had the same plasmid content as −vlsE, but lacked the ospC ORF. −vlsE and −vlsEΔospC were transformed by electroporating with pBSV2GospC (referred to as pCpospC), a shuttle vector carrying the ospC gene and its own promoter .The pC 3 and 4 , from WTpvlsE2 shuttle vector, expressing and transporting VlsE under the control of the ospC promoter and carrying the ospC signal sequence, was constructed as follows. The region of cp26 from nucleotides 16647–16971, encompassing the ospC promoter and ospC signal sequence, was amplified from WT gDNA with Vent polymerase using primers 1 and 5 (ospCp(+ss). The vlsE locus was amplified without the predicted signal sequence from pCpvlsE1 with Vent polymerase using primers 6 and 7 (ospCp(+ss), yielding pBSV2G-ospCp(+ss)-vlsE (pCpvlsE2).The pC 1 and 5 , and lig 6 and 7 , and ligpvlsE1 shuttle vector, constitutively expressing vlsE from the flaB promoter, was constructed as follows. The flaB promoter was amplified from WT gDNA with Vent polymerase using primers 8 and 9 .The pF 8 and 9 , and lig3 and 10 , and ligpospA shuttle vector, expressing ospA under control of the ospC promoter, was constructed as follows. The region of lp54 from nucleotides 9457 to 10278, encompassing the ospA ORF, was amplified from WT gDNA with Vent polymerase using primers 11 and 12 .The pC1 and 12 , and ligpospA shuttle vector, constitutively expressing ospA from the flaB promoter, was constructed as follows. The region of lp54 from nucleotides 9457 to 10278, encompassing the ospA locus, was amplified from WT gDNA with Vent polymerase using primers 11 and 12 .The pF1 and 12 , and ligospC strains  with SacI-NotI digested JK109. A plasmid with the correct fusion was named pOVK.To make a plasmid for replacing the rimers 2  and JK17 et al., . We ampl3 and 13 . Both ofospC promoter-vlsE ORF fusion was moved into B. burgdorferi by transforming WT bacteria with approximately 5 μg of methylated pOVK . C3H/HeN and C3H/HeN SCID mice were purchased from Harlan Sprague-Dawley . RML mice are outbred, derived from Swiss-Webster mice, and bred at the Rocky Mountain Laboratories. IRW mice are inbred, derived from RML mice, and also bred at the Rocky Mountain Laboratories. For infection studies, mice were injected with 80% of the inoculum intraperitoneally and 20% of the inoculum subcutaneously. In some cases (as indicated in the Results), bacteria were inoculated into the intradermal-subcutaneous compartment, with the appropriate number of bacteria provided in a single 100 μl injection with a 27 ga needle. The mice were euthanized three to six weeks post-inoculation, and ears, bladders, and ankle joints harvested and cultured in 10 ml BSKII medium. Isolates from mice infected with 4 and 15 , to asseBorrelia burgdorferi protein lysates  were separated in 12.5% SDS-polyacrylamide gels and immunoblotted as described (Bestor et al., et al., et al., et al.,


Hearing loss has been linked to many types of cognitive decline in adults, including an association between hearing loss severity and dementia. However, it remains unclear whether cortical re-organization associated with hearing loss occurs in early stages of hearing decline and in early stages of auditory processing. In this study, we examined compensatory plasticity in adults with mild-moderate hearing loss using obligatory, passively-elicited, cortical auditory evoked potentials (CAEP). High-density EEG elicited by speech stimuli was recorded in adults with hearing loss and age-matched normal hearing controls. Latency, amplitude and source localization of the P1, N1, P2 components of the CAEP were analyzed. Adults with mild-moderate hearing loss showed increases in latency and amplitude of the P2 CAEP relative to control subjects. Current density reconstructions revealed decreased activation in temporal cortex and increased activation in frontal cortical areas for hearing-impaired listeners relative to normal hearing listeners. Participants' behavioral performance on a clinical test of speech perception in noise was significantly correlated with the increases in P2 latency. Our results indicate that changes in cortical resource allocation are apparent in early stages of adult hearing loss, and that these passively-elicited cortical changes are related to behavioral speech perception outcome. Adults with hearing impairment have been shown to exhibit concomitant deficiencies in cognitive performance , suggesting that increases in auditory deprivation may subsequently influence overall cognitive decline Lin, ,b. Lin eIn this study, we examined cortical re-organization resulting from HL in adult listeners with mild-moderate sensorineural hearing impairment using high-density EEG. We evaluated obligatory, passively-elicited P1, N1, and P2 components of the cortical auditory evoked potential (CAEP) using source localization. We correlated CAEP changes with performance on a clinical test of speech perception in noise to better understand the impact of cortical changes in early stages of hearing decline.n = 17). Subjects were recruited using fliers and recruitment letters. Consent was obtained through documentation approved by the University of Colorado at Boulder Institutional Review Board. Hearing acuity was measured using standard clinical audiometric procedures. Normal hearing (NH) thresholds [below 25 dB Hearing Level (HL)] for frequencies ranging from 0.25–8 kHz were observed for eight of the participants , while the remaining nine demonstrated HL . The HL group showed, on average, NH from 0.25 through 1 kHz and a mild-to-moderate sensorineural HL bilaterally from 2 to 8 kHz. Mean threshold audiograms for the two groups are shown in Figure t(15) = −1.69, p = 0.537].Adults between the ages of 37 to 68 years participated in this study n = 7. SubjecThe QuickSIN™, a clinical measure of auditory threshold for sentences in background noise, was used to determine acuity of speech perception in background noise  and seated in a reclining chair in an electro-magnetically shielded sound booth. Auditory stimuli were presented via stimulus software E-Prime 2.0. The recording sampling rate was 1 kHz, with a band-pass filter of 0.1–200 Hz.EEG topographic map analysis was completed offline using Net Station 4 . A two-dimensional voltage map was generated for each group grand average waveform for each of the three obligatory CAEP peak components . Regions of interest (ROI) were identified based on the greatest group differences for each CAEP component. Four ROIs were determined to be present: the frontal region, central region, the left frontal hemisphere (LH), and the right frontal hemisphere (RH). Individual EEG data was then exported from Net Station and imported into the EEGLAB toolbox  is a statistical procedure that estimates a focal CDR with zero localization error using actual source and measurement variance , and right frontal hemispheric regions (RH). Three obligatory CAEP components elicited by the speech sound were evaluated: the P1 (occurring at approximately 70 ms), N1 (at approximately 100 ms), and P2 (at approximately 180 ms). Group differences for the amplitude and latency of each component were analyzed using a One-Way ANOVA, and planned F = 8.7, p = 0.005], the central ROI , and the LH ROI , but not at the RH ROI . P2 latency was found to be significantly longer for the HL group in the frontal ROI , but not the central , LH , or RH  ROIs. P1 amplitude did not differ significantly between groups in any ROI . P1 latency showed no significant difference . Similarly, the N1 component did not differ significantly between groups in amplitude , or latency . Figure P2 amplitude was found to be significantly larger in the HL group (relative to the NH group) for the frontal ROI [It should be noted that we presented the auditory stimuli at a comfortably loud conversational level for our participants. The /ba/ stimulus is comprised of spectral energy occurring mainly in the low-mid frequency region 0.5–2 kHz) . This difference in performance has been found in similar studies with NH listeners and listeners with HL , suggesting that increases in P2 latency were associated with greater difficulty in perceiving speech in noise. We did not see a significant correlation between QuickSIN™ scores and P2 amplitude.QuickSIN scores were correlated with P2 latency and amplitude. All participants were included in the correlation, as HL can be considered a gradual decrease in threshold starting at 0 dB HL. Frontal ROI P2 latency showed a significant positive correlation with speech performance in background noise . Thus, as HL increased across participants, there was a corresponding increase in P2 amplitude. No significant correlation was observed between P2 latency and high-frequency PTA.Frontal P2 amplitude showed a significant positive correlation with high frequency Pure Tone Average (PTA), i.e., the degree of hearing impairment at 2–8 kHz for both ears  increased P2 CAEP amplitude and latency, (2) reduced activation in temporal auditory cortical regions, (3) activation of frontal cortical regions in response to auditory stimulation, (4) significantly poorer speech perception in background noise that correlated with increased P2 latency and (5) a significant correlation between increased P2 amplitude and hearing thresholds at high frequencies . Thus, even in relatively early stages of HL and early stages of auditory processing, adult subjects appear to show significant alterations in cortical activation.Our finding of increased P2 amplitude for HL listeners is consistent with previous studies, which documented increased P2 CAEP amplitude in older adults who were long-time hearing aid users  relative to NH listeners Figure  and showIt is surprising, however, that we observed that frontal cortical regions, typically associated with cognitive processing, were engaged in response to a passive auditory task that did not require the participants' attention. This finding suggests that compensatory processing may begin at early stages of central auditory processing in adult-onset HL . The nature of this plasticity is observed as a re-allocation of cortical resources from temporal auditory areas to frontal cognitive areas, which appear to be recruited to assist with processing of auditory stimuli even at the level of passive listening. Overall, our results are consistent with the hypothesis that HL appears to initiate a process of resource re-allocation, which results in increased cognitive load (Pichora-Fuller et al., Our results demonstrate auditory cortical re-organization in the form of decreased temporal activation and increased frontal activation in early stage HL of mild-moderate severity using passively elicited EEG responses. Furthermore, increased latency and amplitude of the P2 component were associated with decreases in speech perception performance and increase in hearing threshold, respectively. Due to the strong relationship between HL and cognitive deficits, such as dementia, that arise later in life, it is important that clinical evaluation of cognitive reserve in HL be included as part of intervention services. Future research should focus on better understanding the relationship between the severity of cognitive re-allocation in relation to severity of HL as well as reversibility of re-organization as a result of intervention with amplification.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.


The murine retina has the great advantage that the effect of gene inactivation for Ni2+-sensitive Ca2+ channels can be analysed to prove or disprove that any of these Ca2+ channels is involved in retinal signalling.Light-evoked responses from vertebrate retinas were recorded as an electroretinogram (ERG). The b-wave is the most prominent component of the ERG, and in the bovine retina its NiCl2+-sensitive voltage-gated Ca2+ channels were used to record their ex vivo ERGs.Superfused retinas from different murine genotypes lacking either one or both highly Niv2.3 R-type or Cav3.2 T-type or both voltage-gated Ca2+ channels were superfused with a NiCl2 (15 μm) containing nutrient solution. The change in the b-wave amplitude and implicit time, caused by NiCl2, was calculated as a difference spectrum and compared to data from control animals. From the results, it can be deduced that Cav2.3 contributes rather to a later component in the b-wave response, while in the absence of Cav3.2 the gain of Ni2+-mediated increase in the b-wave amplitude is significantly increased, probably due to a loss of reciprocal inhibition to photoreceptors. Thus, each of the Ni2+-sensitive Ca2+ channels contributes to specific features of the b-wave response.The isolated retinas from mice lacking Ca2+-sensitive Ca2+ channels contribute to transretinal signalling. Based on the results from the double knockout mice, additional targets for NiCl2 must contribute to transretinal signalling, which will be most important for the structurally similar physiologically more important heavy metal cation Zn2+.Both high-affinity Ni In addition, Cav2.2-specific immunoreactivity was found in the outer and inner nuclear layers. But staining for Cav2.3 (R-type) Ca2+ channels was rather widely distributed in all three nuclear layers and in the inner plexiform layer (2+ currents (L-rich) and those expressing various T-type Ca2+ currents (T-rich cone bipolar cells)  .2+ channel regulation in horizontal cells may be important for shaping graded potentials derived from photoreceptors and transferred to bipolar neurons , the b-wave. ON-centre bipolar and horizontal cells are believed to be the major contributors to the b-wave in addition to third-order neurons as amacrine cells (for review of literature see  neurons .2+ channels consist of an ion-conducting α1 subunit and a set of auxiliary subunits (for review see: (v1), non-L-type (Cav2) and T-type (Cav3) voltage-gated Ca2+ channels, according to their electrophysiological and pharmacological properties. P/Q-, N- and E/R-type channels are members of the non-L-type channels, and their α1 subunits are addressed as Cav2.1, Cav2.2 and Cav2.3, respectively  and in the auxiliary β2-subunit cause the loss of visual sensitivity . They aectively . Transcrn so far . Low-volt retina . Particue retina , as mutasitivity , and mutlindness , but alslindness .2 caused biphasic effects. While low NiCl2 concentrations (up to 30 μm) stimulated the b-wave response, high concentrations (100 μm) reduced the b-wave response  is known to modulate neuronal signal transduction. Therefore, we switched to investigations of the murine retina. For both Ca2+ channel types (Cav2.3 and Cav3.2), nonlethal mouse lines are available . All stock solutions for the nutrient solutions were sealed in glass tubes  to establish reproducibility of ERG recordings. Deionized water (<0.1 μS/cm) was additionally glass distilled and autoclaved in glass bottles. All solutions were prepared in autoclaved glass bottles, using sterile distilled water.For the preparation of the murine retina , a superMurine retinas were isolated from mice of our animal facility department, in which the light–dark regime was 12:12 hr, and the light intensity between 5 and 10 lx at the surface of the animal cages. The mice were preincubated for dark adaptation overnight.v2.3-  and Cav2.3(−|−), each after the fourth backcrossing in C57Bl/6 mice. For Cav3.2, mouse lines were used in pure C57Bl/6 background. For the experiments, we used littermates, and genotyped by RT-PCR.DNA-containing tissue samples were collected from tail biopsies. DNA was extracted and used as template for genotyping. Transcripts of Ca3 region . For CavThe German Animal Experiments Inspectorate approved the treatment of mice involved in the experiments and also approved the animal-housing facilities. We have considered the ethical aspects of the study and followed the guidelines of the European Communities Council Directive of 24 November 1986 (86/609/EEC).m NaCl, 2 mm KCl, 0.1 mm MgCl2, 0.15 mm CaCl2, 1.5 mm NaH2PO4, 13.5 mm Na2HPO4 and 5 mm glucose. The resulting pH was 7.8. The bathing solution was empirically optimized  containing 120 mptimized . Subsequptimized . Initialptimized  were perEnucleated murine eyes were opened from the front side first by punching a tiny hole through the cornea to relieve the aqueous humour from the anterior and posterior chamber of the eye and thus to reduce the tension. Thereafter, the cornea was removed, and a tiny triangle-shaped incision was made into the sclera for later separation of the proximal eye cup, and for the immediate introduction of small forceps. Iris and lens were carefully removed. Consecutively, the opened eye cup was held by forceps to detach the retina from the pigment epithelium by repetitive moving in the nutrient solution. The successive separation of the retina from the pigment epithelium has to be performed gently and is supported by the cutting of the sclera layers from outside.Following dissection from the underlying pigment epithelium, the retina was mounted on a plastic mesh occupying the centre of the perfusing chamber. The retina segment adheres by itself, and the supporting ring is turned upside down to move the retina-loaded ring into the holder of the recording chamber. The recording chamber, which is otherwise identical to the former chamber for recording of bovine retina segments , is lockvia two silver/silver chloride electrodes on either side of the retina with the recording chamber containing the retina placed in an electrically and optically isolated air thermostat. The recording chamber was continuously perfused at a perfusion velocity of 2 ml/min, controlled by a roller pump. Temperature was kept constant at 27.5°C. To avoid hypoxic conditions during the preparation of the isolated retinas as well as during the perfusion, the nutrient standard solution was pre-equilibrated and continuously saturated with oxygen and sometimes monitored by a Clark oxygen electrode during the experiments. From the dark-adapted retina, ERGs in response to a single white flash were recorded at intervals of 3 min. Equilibrium was reached between 10 and 30 min of perfusion for the murine retina. The duration of light stimulation was 500 ms controlled by a timer operating a mechanical shutter system. The prestimulus delay was 150, 250 or 350 ms as indicated by the stimulus bar in each figure. The flash intensity was set to 63 mlx at the retinal surface using calibrated neutral-density filters. The ERG was amplified and bandpass-limited between 1 and 300 Hz. The signal was AD converted and stored using a PC-based signal acquisition and analysis system (DASY-Lab).The ERG was recorded For each experiment, an unused retina segment or full retina was transferred to the recording chamber. The retina was superfused with the nutrient solution and stimulated repetitively until stable responses were recorded. Switching from one solution to another was performed without compensation for the latency of drug delivery, which can be seen in the labelled bars in each figure.The a-wave amplitude was measured from the zero line to the consecutive trough. The b-wave amplitude was measured from the trough of the a-wave to the peak of the b-wave, as indicated. For the statistical analysis of the a- and b-wave amplitude, the software programs ‘Origin 6.0’  and ‘Excel 2003’ (Microsoft) were used. ERG traces were generated from central moving average values, for which the unweighted mean of 13 data points was determined (from six previous to six future data points).t-test. Levels of p ≤ 0.05 were considered as statistically significant (*) and p ≤ 0.01 as highly significant (**).Significance was calculated with Student’s 2 (15 μm) up to 1.5-fold  to 69.8 ±1.4 μV (n = 8), which was partially reversed to 55.7 ± 1.2 μV (n = 8) after washout of NiCl2. During this procedure, the implicit time was severely shortened  in Cav2.3-deficient mice was a statistically significant (p = 0.034) difference from the controls of a 1.41-fold increase  and a temperature of only 27.5°C instead of 30°C . Under tm NiCl2  as knownm NiCl2 . Finallyhortened . The shihortened , which wmplitude . In a tomplitude . In a sicontrols . If we qe retina .2-mediated stimulation in the retinas from control mice in more than 50% of the experiments. Therefore, we shifted the isolation protocol to a version with no prolonged preincubation period, taking into account lower initial b-wave amplitudes. The freshly isolated retina segments were immediately placed into a recording chamber, and the recording protocol was started to reach a rather stable incomplete ERG with no (n = 5) yielded a-waves with an amplitude of −79 ± 17 μV (v2.3-deficient mice (n = 6) did not differ significantly from controls with −111 ± 29 μV (p = 0.40). However, after inactivation of Cav3.2 (n = 4 retinas) and after inactivation of both highly Ni2+-sensitive Ca2+ channels (n = 6), a-wave amplitude was increased significantly to −212 ± 18 μV (p = 0.0011) and −184 ± 39 μV (p = 0.049), respectively, suggesting that Cav3.2 rather than Cav2.3 may be involved in reciprocal signalling to photoreceptors. No significant differences were found for implicit times between any of the four genotypes  superfusion . The mean ERGs after NiCl2 superfusion  were compared for the four genotypes by its implicit times (n = 5 retinas) for control mice and was significantly reduced in Cav2.3-deficient mice to 346 ± 12 ms , confirming the observation from the visual inspection of the overlay traces  and 480 ± 21 ms (n = 6), respectively, and did not differ significantly from control mice, suggesting that at least in Cav2.3-deficient mice a major component of the b-wave is missing, which in control animals causes the delayed depolarization during retinal signalling.The normalized Niit times . The meay traces . The cor2+-induced component of the b-wave was also compared between the four genotypes. It is significantly increased in Cav3.2-deficient mice  and double knockout animals  compared to controls  and Cav2.3-deficient mice (70 ± 7 μV n = 14)  , suggest2+-triggered neurotransmitter release in vertebrate photoreceptors and bipolar cells of the retina is mediated by high- and low-voltage-gated Ca2+channels  NiCl2 solutions. For the bovine retina, a similar amplitude increase was observed when superfusing ZnCl2   , one may2+-mediated stimulation was lost faster than the basal transretinal signalling. The slow but continuous increase in the b-wave amplitude must be interpreted as a continuous loss of GABAergic inhibition onto ON-bipolar cells, which mainly contribute by their light-evoked depolarization to the ERG b-wave. It was difficult to standardize this slow reduction of physiological inhibition. Therefore, any preincubation of the isolated retina was shortened to a minimum, just to reach a reliable starting condition for ERG recording. To calculate the ‘gain’ of Ni2+-mediated b-wave increase, the maximum difference in amplitude was compared between four genotypes, and the normalized shape of the ERG was analysed and compared as a difference in implicit time. All these precautions were necessary to get a reproducible way of experimental work for answering three main questions. First, does Cav2.3 in mice contribute to the well-described NiCl2-mediated increase in the ERG b-wave amplitude as observed from the isolated and superfused bovine retina . As mentioned, the accuracy was reduced after such a long 4-h preincubation period. Therefore, we switched to short pre-equilibration (30 min) and calculated differences for the NiCl2-mediated effects after an additional 30-min superfusion of 15 μm NiCl2. Even after such an application protocol, the NiCl2-mediated effects on the amplitude did not reach the level of significance, but the shape of the NiCl2-induced component was different in Cav2.3-deficient mice compared with controls, leading to a significantly shortened implicit time  on the calculated increase in the ERG b-wave amplitude  GABAergic signalling as it was analysed for rod bipolar cells in rat  retina, the temporal characteristics of reciprocal signalling were analysed recently more in detail  may change the ERG b-wave response by two different voltage-gated Ca2+ channels and also by additional highly Ni2+-sensitive targets. While Ni2+ cations may present nonphysiological cations, future studies may include the more physiological effects of Zn2+, which is highly concentrated in the vertebrate retina and reveals pronounced effects on the isolated bovine retina (Together, the results from our study and the results reported in the literature provide strong evidence that low NiCle retina . Future e retina  and the e retina .