It is already accepted that telocytes (TCs) represent a new type of interstitial cells in human dermis. In normal skin, TCs have particular spatial relations with different dermal structures such as blood vessels, hair follicles, arrector pili muscles or segments of sebaceous and/or eccrine sweat glands. The distribution and the density of TCs is affected in various skin pathological conditions. Previous studies mentioned the particular (ultra)structure of TCs and also their immunophenotype, miR imprint or proteome, genome or secretome features. As fibroblast is the most common intersitital cell , a dedicated comparison between human skin TCs and fibroblasts (Fbs) was required to be performed. In this study, using different techniques, we document several points of difference between human dermis TCs and Fbs. By transmission electron microscopy (TEM) and scanning electron microscopy (SEM), we demonstrated TCs with their hallmark cellular prolongations – telopodes. Thus, we showed their ultrastructural distinctiveness from Fbs. By RayBio Human Cytokine Antibody Array V analyses performed on the supernatant from separately cultured TCs and Fbs, we detected the cytokine profile of both cell types, individually. Two of 79 detected cytokines – epithelial-derived neutrophil-activating peptide 78 and granulocyte chemotactic protein-2 – were 1.5 times higher in the supernatant of TCs (comparing with Fbs). On the other hand, 37 cytokines were at least 1.5 higher in Fbs supernatant (comparing with TCs), and among them six cytokines – interleukin 5, monocyte chemotactic protein-3 (MCP-3), MCP-4, macrophage inflammatory protein-3, angiogenin, thrombopoietin – being 9.5 times higher . In summary, using different techniques, we showed that human dermal TCs and Fbs are different in terms of ultrastructure and cytokine profile. Furthermore, alterations of normal TCs ultrastructure were described in psoriatic dermis Telocytes (TCs) are already acknowledged as new and distinct type of interstitial cells of human dermis In connective tissue, fibroblasts (Fbs) have mainly structural functionality, being responsible for production of components of the extracellular matrix: collagen and elastic fibres, ground substance and cytokines In this study, we compared TCs and Fbs from human dermis in terms of ultrastructure and cytokine profile. We bring more evidence that in human dermis, TCs and Fbs are two distinct cell population. These data will become a requisite in establishing the details of TCs' functional potential in skin, in health and disease.et al. Biopsies of normal skin were harvested from 10 patients, as previously presented in Manole Human skin Fbs were purchased from Chinese Academy of Science, Kunming Cell Bank . Telocytes cultures were obtained from human dermal samples taken from male patients accepting foreskin resection. The study was approved by the Ethic Committee of Fudan University . The informed consents were obtained from all of the patients included.3) were processed according to a routine Epon embedding procedure as described previously © Photoshop CS3.Small tissue fragments . Post-fixation was made in 2% osmium tetroxide for 2 hrs. The dehydration was done using a series of increased concentrations of ethanol. The samples were then transferred to a critical point dryer. The cells were then observed and photographed with a JEOL JSW-6390LV  scanning electron microscope, at 5 kV.5/well and maintained at 37°C, in a humidified atmosphere (5% CO2 in air). The cells were continuously cultured for 48 hrs in DMEM , 2 ml for each well, without foetal bovine serum . For further cytokine antibody array measure, the supernatant was collected and stored at −20°C.Human skin Fbs were cultured in the six-well plate  at a density of 1 × 10et al. 3. The fragments were then incubated at 37°C for 4 hrs on an orbital shaker with 1 mg/ml collagenase type II in PBS (Gibco) without Ca2+ and Mg2+. The reaction of collagenase was terminated by 10% FBS (Gibco). Dispersed cells were separated from non-digested tissue by the filtration through a 40 μm diameter cell strainer  and collected by centrifugation at 2000 r.p.m. for 5 min. The cells digested from tissues were plated on 25 cm2 plastic culture flasks in DMEM with 10% FBS (Gibco) and 1% penicillin /streptomycin  at a density of 1 × 105 cells/cm2. The cells were maintained at 37°C, in a humidified atmosphere (5% CO2 in air) until becoming semi-confluent . The culture medium was changed every 48 hrs. In the 5th day, the adherent cells were collected and re-plated into a new six-well culture plate  at a density of 1 × 105/well. The cells were then maintained at 37°C, in a humidified atmosphere (5% CO2 in air), and were continuously maintained in DMEM without FBS (Gibco), 2 ml for each well, for 48 hrs. For cytokine antibody array measure, the supernatant was collected and stored at −20°C. Cells were observed and photographed under a light microscope through 40× objective .Telocytes isolation and cultures were performed in accordance with the method previously described by Hatta Supernatants, obtained from cultured human dermal TCs and Fbs, were analysed with cytokine antibody array . This study was performed by Kangchen Bio-tech Company . Briefly, cytokine array membranes were blocked in blocking buffer for 30 min. and then incubated with samples at room temperature for 1–2 hrs. Samples were then decanted, and membranes were washed. The membranes were incubated with diluted biotin-conjugated antibodies  at room temperature for 1–2 hrs. After the membranes were washed again, 1000-folds diluted horseradish peroxidase-conjugated streptavidin  was added and incubation was continued for 2 hrs at room temperature. The membranes were then washed thoroughly and exposed to detection buffer  in the dark, before being exposed to X-ray film. By comparing the signal intensities, relative expression levels of cytokines could be obtained. The intensities of signals were quantified by densitometry. Biotin-conjugated immunoglobulin G (Cusabio) served as a positive control at six spots to normalize the results from different membranes being compared.P-value for comparisons between two samples, statistical analyses were performed with Student's t-test. The threshold of significance was set at P < 0.05.The SPSS 17.0 software package  was used to process the data. To calculate a 1-3 – running parallel to each other and all being parallel with epidermis basal membrane. Moreover, Tp3 is establishing contacts with the ab luminal surface of capillary endothelial cells.The transmission electron microscopy (TEM) analysis was focused on dermis from healthy donors. Cells with ultrastructure evocative for TCs were recognized in papillary dermis, among other interstitial cells. Commonly, these cells have a small cellular body, containing a large ovoid nucleus rimmed by scarce cytoplasm with rare organelles within, and two visible cellular prolongations A. The ulThe ultrastructural features of TCs are completely different than that of Fbs B. In con1-4) of variable length  shows more evidence on the morphological features of TCs . The typPhase contrast microscopy documented the differences between TCs A and Fbse.g. interleukin 5 (IL-5), monocyte chemotactic protein-3 (MCP-3), MCP-4, macrophage inflammatory protein-3 (MIP-3a), angiogenin, thrombopoietin .A large body of data recorded the presence of Fbs in stroma of various organs. Also, in other organs, the distinctiveness of TCs from different interstitial cells was documented concerning their aspect in cell culture In this study, by TEM and SEM, we documented the distinctiveness of TCs population in human dermis, based on their ultrastructural features and also their peculiar cytokine profile.The ultrastructural characteristics, and also the differences between human dermal TCs and Fbs are in accordance with the previous ones reported in other organs. We found that TCs have a small cell body with Tps – the most particular ultrastructural features of TCs. Tps are very long  and very thin and slender cellular prolongations of unequal calibre, with string of beads appearance. Adversely, Fbs are ultrastructurally featured by a large cell body of various aspects, with short and conical thick cellular prolongations.Furthermore, the individuality of human dermal TCs and their distinctiveness than Fbs was proven using cytokine antibody array. Thirty-nine cytokines (out of 79 detected cytokines) were found to be differentially expressed between TCs and Fbs. These were at least 1.5 times overexpressed either in TCs or Fbs (37 in Fbs and 2 in TCs). Thus, we showed that the expression of these cytokines in TCs is dissimilar to that of Fbs.ab luminal faces of endothelial cells. Furthermore, TCs' positive expression for ENA-78 and also the TEM data showing TCs' close spatiality with mononuclear cells could represent the substrate for the attributed role of TCs in the integration of the intercellular signalling and immune modulation response. However, ENA-78 was credited with contribution in cancer cell proliferation, migration and invasion The expression of ENA-78 and GCP-2 was found to be significantly higher in TCs than in Fbs. ENA-78  is a small neutrophil chemo-attractant cytokine being normally produced as a result of inflammatory stimulation. ENA-78 was also reported to have potential in regulation of angiogenesis Granulocyte chemotactic protein 2 (or CXCL6) is another highly expressed cytokine by TCs. This small cytokine has documented function in granulocytes chemo-attraction Adversely, 37 cytokines were highly expressed in dermal Fbs than in TCs. These cytokines elicit functions in chemotaxis for monocytes  e.g. CXCL 5 – ENA-78 and CXCL-6 – GCP-2) in comparison with Fbs, which have a higher secretion of CC chemokines .The cytokine profiles we identified in the present work for skin TCs and Fbs were different from the results got in the protein profiles comparison between human lung or cardiac TCs and Fbs We may conclude that considering their ultrastructural differences and divergent cytokine profiles, TCs and Fbs are different types of interstitial cells. The peaks of the cytokine expressions of TCs indicate, in skin, their functional roles as regulators of (neo)angiogenesis, and/or immune modulators, probably with implications in skin cancer progression. On the contrary, taking into account their cytokine profile, Fbs denote structural roles and also degrees of involvement in immune response. Our results are preliminary and could constitute a starting point for further studies. Of course, a more diverse spectrum of analyses, also including more cytokines species, would deepen the schism between these two different interstitial cells.


Since the origin of the animal kingdom, marine animals have lived in association with viruses, prokaryotes and unicellular eukaryotes, often as symbionts. This long and continuous interaction has provided ample opportunity not only for the evolution of intimate interactions such as sharing of metabolic pathways, but also for horizontal gene transfer (HGT) of non-metazoan genes into metazoan genomes. The number of demonstrated cases of inter-kingdom HGT is currently small, such that it is not yet widely appreciated as a significant player in animal evolution. Sessile marine invertebrates that vertically inherit bacterial symbionts, that have no dedicated germ line, or that bud or excise pluripotent somatic cells during their life history may be particularly receptive to HGT from their symbionts. Closer scrutiny of the growing number of genomes being accrued for these animals may thus reveal HGT as a regular source of novel variation that can function to extend the host phenotype metabolically, morphologically, or even behaviorally. Taxonomic identification of symbionts will help to address the intriguing question of whether past HGT events may constrain contemporary symbioses. It is increasingly apparent that all sessile marine animals live in intimate association with a diversity of viruses, prokaryotes and unicellular eukaryotes, many of which may function as beneficial symbionts at some or all stages of the animal life cycle . This isQuerying the nature and extent of animal–microbe interactions can increasingly be done at the level of the genome. Genomes clearly reflect the deep, shared ancestry between animals and microbes; all three domains of life share approximately one third of their genes, notably including many that encode central metabolic pathways . Beyond A less broadly appreciated outcome of host–symbiont interactions is the potential through deep evolutionary time for novel functions to be conferred via inter-kingdom horizontal  gene transfer (HGT), broadly defined as “non-genealogical transmission of genetic material from one organism to another” . AlthougHorizontal gene transfer could be another way for symbionts to significantly affect host morphology, physiology, or even behavior, in manner akin to the ‘extended phenotype’ proposed by In recent years, the number of confirmed cases of HGT into animal genomes has been steadily growing as interest is rekindled and growing databases of genomic resources make evidence more robust  phylogenetic evidence that the candidate gene is more closely related to foreign than to animal genes; (ii) genome data showing the candidate gene assembles into a contiguous stretch of DNA with neighboring genes unambiguously of animal origin; and (iii) gene sequence revealing metazoan-like compositional traits, including presence of introns, GC content and codon usage. The second of these requires, of course, the availability of a sequenced and assembled animal genome; the more complete the assembly, the more confident the HGT identification. Where possible, gene expression data showing active transcription of candidate genes in animal cell nuclei can enormously strengthen a case, and also addresses the issue of whether or not the HGT-acquired gene is active in its new genomic context, as discussed below.Wolbachia parasites of arthropods and nematodes – have produced several cases of strong evidence for HGT, but interestingly only some of which have resulted in functional genes in the recipient animal genome  the sea anemone yoxylate  and shikyoxylate  pathwaysase gene ; and  th enzymes ; and  putatively transferred symbiont . This, oHydra; Nematostella as HGTs from bacteria can be traced phylogenetically to a Tenacibaculum-like (Flavobacteria) origin; Nematostella appears to host a bacterial symbiont of the same taxonomy, which is present even in early life history stages (Our ability to address this question depends upon (i) the comprehensiveness of bacterial genome databases that permit assignment of HGTs to a specific taxonomic source, and (ii) knowledge of the taxonomy of contemporary symbionts of the recipient animal host. Data in both of these areas is growing rapidly, so that any patterns that do exist should emerge within the next few years. At present, for many of the demonstrated cases of HGT described above, the taxonomic source of the transfer is currently unknown. One reason for this, as exemplified by the biomineralization gene transferred into sponges , might by stages .Wolbachia, which have resulted in multiple HGT events  and thus how long it has persisted. This returns to the question of longevity as demonstration of genuine integration into the recipient genome, and is best served by HGTs for which evidence in multiple animal lineages permits tracing back to a last common ancestor. Second, it addresses whether animals may be choosy in their source of HGT, even if only as a consequence of accessibility to bacterial DNA. If the phylogenetic source of HGT-acquired genes reflects taxonomy of contemporary symbionts, might this suggest constraint on associations? Could HGT events resulting from symbioses deep in the evolutionary past constrain the taxonomic associations possible in the ecological present? Symbioses between arthropod and nematode hosts and their alpha-proteobacterial T events , will noGiven the temporally very long, and spatially very close, associations between marine invertebrate animals and bacteria, it is entirely feasible that HGT from bacterial to marine animal genomes is much more prevalent than currently appreciated. That marine animal–bacterial symbioses are both ubiquitous and simultaneously exclusive strongly suggests that host and symbionts carefully choose their partners, rather than relying on random associations . Might bIn insects and vertebrates in particular, evidence is mounting for anatomical, cellular, and molecular determinants that act during early development to prepare progeny for their lifetime of interactions with microbes (reviewed in The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The eosinophil-derived neurotoxin (EDN/RNase2) and its divergent orthologs, the mouse eosinophil-associated RNases (mEars), are prominent secretory proteins of eosinophilic leukocytes and are all members of the larger family of RNase A-type ribonucleases. While EDN has broad antiviral activity, targeting RNA viruses via mechanisms that may require enzymatic activity, more recent studies have elucidated how these RNases may generate host defense via roles in promoting leukocyte activation, maturation, and chemotaxis. This review provides an update on recent discoveries, and highlights the versatility of this family in promoting innate immunity. EDN, anntration . Recent ntration ,5,6,7, cntration , eosinopntration , and vacntration . This rentration ,13,14,15Gordon  was the In 1986, Gleich and colleagues  publisheSaguinus oedipus) and the Northern owl monkey (Aotus trivirgatus). While the EDN orthologs from these primates include the crucial cysteines and catalytic components, and likewise maintain 73% and 71% amino acid sequence similarity to human EDN, respectively, enzymatically, their turnover numbers (kcat) were reduced by a factor of about 100. Structure/function analysis indicated that divergence near the C-terminus led to this discrepancy, and that penultimate arginine and/or isoleucine residues were necessary to support the elevated level of enzymatic activity observed in the human ortholog. The constraints promoting divergence in this lineage and among all RNase A-type ribonucleases are a subject of significant and ongoing interest [Sequences directly orthologous to human EDN and human ECP were readily identified in higher primate genomes, although the degree of inter-species divergence was notably high for otherwise functional coding sequences ,32. Inteinterest ,27,36,37The human genome includes only one pseudogene of the EDN/RNase 2 lineage . This seZhang and Rosenberg  characteMus musculus encodes a cluster consisting of numerous functional mEar genes and pseudogenes that have diverged via a mechanism characterized as rapid birth-death and gene sorting ; ,62). Anti.e., TLR2, TLR4, NOD1 and NOD2), although it is not yet clear how expression of these receptors contributes specifically to eosinophil function in health and disease. Although EDN, among the four granule proteins, does not display broad-spectrum anti-microbial activity [Clostridium difficile, in contrast to a probiotic strain, such as Bifidobacteria, which did not induce EDN release. In a subsequent study, eosinophils again released EDN selectively, notably in response to provocation with Staphylococcus aureus, but not Hemophilus or Prevotella species [Human eosinophils express a substantial range of pattern recognition receptors . Among activity ,65, it iactivity  reported species . The wayYang and colleagues ,69,70 ha+progenitor-derived dendritic cells generated in tissue culture; migration was pertussis-toxin sensitive although the receptor has not yet been identified [+ DCs (among other cells) into subcutaneous air spaces in experiments performed in BALB/c mice  responded in a manner that was indistinguishable from their wild-type counterparts. Expression of cell surface markers and cytokine release remains to be examined , as well as the potential to harness these observations toward future therapeutic goals. Likewise in need of further clarification are nature, specificity and outcome of the interactions of EDN and potentially the mEars with PRRs. While EDN has been shown to interact with TLR2, future studies may extend these observations to include additional PRRs and likewise may encompass one or more orthologous mEars. Finally, it will be crucial to understand what regulates expression of EDN and mEars in cells other than eosinophils, notably in response to specific classes of proinflammatory mediators. The unique responses of mEar 11 and its expression in macrophages in response to Th2 cytokines and IL-33 may be just the first of a larger series of similar observations. While many questions remain unanswered, the research featured in this review highlights the versatility of EDN and the mEars and the numerous ways in which these RNase A-type ribonucleases may promote host defense.The RNase A-type ribonucleases, and EDN in particular, remain intriguing subjects for future study. Among the issues that remain to be addressed, we still do not have a clear understanding of enzymatic activity and its role in biological function. Thus, while the antiviral activity of EDN is directly dependent on active ribonuclease activity, these experiments have for the most part been carried out in tissue culture, and need to be explored further in


Tinnitus is the perception of phantom sound without an external auditory stimulus. Using neuroimaging techniques, such as positron emission tomography, electroencephalography, magnetoencephalography, and functional magnetic resonance imaging (fMRI), many studies have demonstrated that abnormal functions of the central nervous system are closely associated with tinnitus. In our previous research, we reported using resting-state fMRI that several brain regions, including the rectus gyrus, cingulate gyrus, thalamus, hippocampus, caudate, inferior temporal gyrus, cerebellar hemisphere, and medial superior frontal gyrus, were associated with tinnitus distress and loudness. To reconfirm these results and probe target regions for repetitive transcranial magnetic stimulation (rTMS), we investigated the regional cerebral blood flow (rCBF) between younger tinnitus patients (<60 years old) and the age-matched controls using single-photon emission computed tomography and easy Z-score imaging system. Compared with that of controls, the rCBF of tinnitus patients was significantly lower in the bilateral medial superior frontal gyri, left middle occipital gyrus and significantly higher in the bilateral cerebellar hemispheres and vermis, bilateral middle temporal gyri, right fusiform gyrus. No clear differences were observed between tinnitus patients with normal and impaired hearing. Regardless of the assessment modality, similar brain regions were identified as characteristic in tinnitus patients. These regions are potentially involved in the pathophysiology of chronic subjective tinnitus. Tinnitus is the perception of phantom sound or noise in the absence of a real external auditory stimulus –4. With The etiology of tinnitus is debated between peripheral pathology, including cochlear and auditory nervous damage vs. dysfunction of the central nervous system (CNS) . Recent Single-photon emission computed tomography (SPECT) is a functional neuroimaging method that can be used with 99 m Tc-ethylcysteinate dimer (99 m Tc-ECD) as a tracer to estimate regional cerebral blood flow (rCBF) . SeveralThe easy Z-score imaging system (e-ZIS) for SPECT was developed by Matsuda H et al. –27 and iThis study intended to reconfirm the results of our previous resting-state fMRI study using a different modality and method. We used SPECT and e-ZIS to compare rCBF between young/middle-aged tinnitus patients and controls. This study also intended to identify therapeutic target regions for repetitive transcranial magnetic stimulation (rTMS). Previous studies have used the primary auditory cortex as the target of rTMS . Our attSeventeen young and middle-aged subjects (25–59 years old) were re-enrolled in this study with written consent. The subjects were outpatients (August 2013–May 2015) at the clinic of the Department of Otolaryngology-Head and Neck Surgery at Wakayama Medical University Hospital. This study was conducted according to the Declarations of Helsinki with the approval of the Wakayama Medical University Ethics Committee (No. 1265). The subjects  did not have a history of seizures or a suspected diagnosis of organic brain damage, brain tumors, or psychiatric diseases (including major depressive disorder) prior to the onset of tinnitus. No patients received medication with antianxiety, antidepressant, anti-epileptic, or other psychotropic drugs. Tinnitus-related distress was assessed using the standard tinnitus handicap inventory (THI) . StatistExpert otorhinolaryngologists made the determination of normal middle ear status. Hearing levels of subjects were determined using a pure-tone audiometer . Subjects were presented with pure tones ranging from 250 Hz to 12 kHz to each ear until the detection threshold was reached. The number of subjects with normal hearing (hearing level in any frequency <30 dB) was 6, while that with mild to severe hearing loss was 11.Relative CBF was estimated using the 99 mTc-ECD-SPECT as previously described –27, 29. Methods used were based on previously-reported protocols . We usedThe Z-scores at the peak voxel in several brain regions were compared between subjects with normal and impaired hearing using Student’s t-test with MATLAB software .Patients were divided into two groups based on their hearing acuity as follows: normal hearing, hearing level in any frequency <30 dB; impaired hearing, mild to severe hearing loss .The distribution pattern of Z-scores in Regardless of hearing acuity, tinnitus patients exhibited alterations in rCBF in the bilateral medial superior frontal gyri, bilateral cerebellar hemispheres, cerebellar vermis, bilateral middle temporal gyri, bilateral fusiform gyri, and bilateral middle occipital guri. These regions were almost identical to those associated with tinnitus symptoms as revealed by resting-state fMRI .The commercially provided control database does not include audiological information; thus, might include subjects, especially in aged subjects with mild hearing loss and tinnitus. However, few subjects in the young and middle-aged control group are likely to have hearing loss and tinnitus. Therefore, we examined differences in rCBF between young and middle-aged, medication-free patients compared with age-matched controls.rGCa and tinnitus loudness was also detected in the posterior cingulate gyrus, while hypoperfusion was not detected in any tinnitus patients. As hypoperfusion in the posterior cingulate gyrus is diagnostically significant in very early Alzheimer’s disease [Significant hypoperfusion was observed in the bilateral medial superior frontal gyrus, where a significant negative correlation was observed between the regional global connectivity  and tinnitus loudness . This ne disease –27, our  disease –34. ThesrGCa and tinnitus distress in the right inferior temporal gyrus. A significant positive correlation between rGCa and tinnitus loudness was observed in the bilateral hippocampus. These studies emphasize the contribution of the lower temporal lobes, including the amygdala, hippocampus, parahippocampus, and fusiform gyrus, to tinnitus perception.Hyperperfusion was observed in the bilateral middle temporal gyri and right fusiform gyrus. This result was similar to a recent report that the left parahippocampus was significantly hyperperfused in tinnitus with normal hearing . In our rGCa and tinnitus distress [Remarkable hyperperfusion was also observed in both cerebellar hemispheres and cerebellar vermis. In the right cerebellar hemisphere, a significant positive correlation was observed between distress . Severaldistress –37. The distress . The posdistress , 37. Fundistress , 39. A rdistress . Surgicadistress . TogetherGCa in the primary auditory cortex. A recent study using PET also showed that the activity in the primary auditory cortex did not differ between tinnitus patients and controls [Many studies have indicated the involvement of the primary auditory cortex (Heschl gyrus) in tinnitus. Elevated metabolic activity in the left primary auditory cortex has been reported . This hycontrols . These rThe commercially-provided control database might include patients with mild hearing loss and/or tinnitus, although these controls were healthy and neurologically normal. In this study, we compared young (age <40 years) and middle-aged (age 40 ~59), medication-free subjects with those of the normal control datebase  provided by Fuji Film because it is unlikely that these control subjects had audiological disorders. However, it should be careful to conclude that these regions are specific to tinnitus itselfWhether the altered rCBF in these regions is causative of tinnitus or secondary or compensatory changes in response to tinnitus remain unclear. Changes in rCBF may be affected by age, for which we corrected using a corresponding age-matched control group. Ambient noises during image acquisition of SPECT may affect the resting-state rCBF depending on the hearing acuity of patients. However, hearing acuity did not influence the change in rCBF in tinnitus. The laterality and duration of tinnitus could not be taken into consideration in this study. These factors may affect the laterality and degree of rCBF in tinnitus.rGCa and tinnitus symptoms. In addition, these regions may be possible targets for tinnitus treatment.Regardless of hearing acuity, rCBF in tinnitus patients was lower in DMN regions, such as the bilateral medial superior frontal gyri, and higher in memory and emotional networks, such as the bilateral lower temporal lobe, compared with that in normal controls. Hyperperfusion was also observed in the bilateral cerebellar hemispheres and vermis. These regions were almost identical to those exhibiting an association between the S1 DataThe name of each NIFTI file indicates patient number as shown in (ZIP)Click here for additional data file.S1 List(ZIP)Click here for additional data file.


The aim was to describe the outcome of neonatal hearing screening (NHS) and audiological diagnosis in neonates in the NICU. The sample was divided into Group I: neonates who underwent NHS in one step and Group II: neonates who underwent a test and retest NHS. NHS procedure was automated auditory brainstem response. NHS was performed in 82.1% of surviving neonates. For GI, referral rate was 18.6% and false-positive was 62.2% . In GII, with retest, referral rate dropped to 4.1% and false-positive to 12.5%. Sensorineural hearing loss was found in 13.2% of infants and conductive in 26.4% of cases. There was one case of auditory neuropathy spectrum (1.9%). Dropout rate in whole process was 21.7% for GI and 24.03% for GII. We concluded that it was not possible to perform universal NHS in the studied sample or, in many cases, to apply it within the first month of life. Retest reduced failure and false-positive rate and did not increase evasion, indicating that it is a recommendable step in NHS programs in the NICU. The incidence of hearing loss was 2.9%, considering sensorineural hearing loss (0.91%), conductive (1.83%) and auditory neuropathy spectrum (0.19%). Neonatal intensive care units (NICU) have experienced great development in the last 20 years. The mortality rate of high-risk newborn infants has gradually decreased as medical science has advanced. In Brazil, recent data revealed increased incidence of prematurity and low birth weight. One of the most important health indicators that showed improvement was the reduction in infant mortality rate . ConsequIt is known that the same conditions responsible for the deaths during the neonatal period are the most important causes of illness of the neonates. Neonatal morbidity associated with particularly severe asphyxia, severe infection, congenital anomalies, and severe respiratory distress results in delayed death or serious sequels . NewbornMany studies have reported that damage caused by hearing loss in infancy is often irreversible, affecting not only the development of speech and language but also the cognitive, intellectual, cultural, and social child development . Early aUniversal neonatal hearing screening (UNHS) is the first step in a neonatal hearing health program. It must be followed by multidisciplinary care for diagnosis and should be started as an early intervention process with the use of personal amplification devices and communication habilitation.Risk factors associated with hearing loss were recommended by the Joint Committee on Infant Hearing (JCIH) in 2007 . The useJCIH also recommended the use of physiological procedures for screening . For neonates without risk factor for hearing loss (RFHL), any of the methods are considered appropriate. However, for neonates with RFHL and especially those who remained in the NICU, the use of AABR is indicated, considering the higher occurrence of retrocochlear losses, such as the auditory neuropathy spectrum disorder that cannot be identified when using EOAE.For the diagnosis of hearing loss, a protocol with electrophysiologic measures, EOAE, tympanometry, and acoustic reflex testing is recommended. This battery of tests is critical to determine the type, degree, and configuration of hearing loss and to guide auditory rehabilitation .The Woman's University Hospital (CAISM) at State University of Campinas (UNICAMP) is a teaching hospital that is considered the largest hospital of attention to women's health in the state of São Paulo, Brazil. The CAISM is prepared to receive high-risk pregnancies by maternal and/or fetal diseases of an extensive region of São Paulo totaling more than 60 counties. The hearing screening procedures performed at this hospital have been modified over the years. From the start, the hearing screening program for newborns admitted to the NICU used AABR, whenever it was possible, prior to discharge, in a single step. In 2011, new equipment was acquired and in 2012 the retest was introduced. Currently, it is important to evaluate the results obtained by the program in order to support its implementation. Thus, the objective of this research was to evaluate the outcome of the results in neonatal hearing screening program and audiological diagnosis in neonates who were hospitalized in the neonatal intensive care unit.This is a clinical, prospective, cross-sectional study. The institutional review board approved this study (Protocol no. 1085/2009).The inclusion criteria were neonates who remained in the NICU from CAISM/Unicamp for at least 48 hours, from March 2011 to April 2013.The exclusion criteria were neonates who stayed less than 48 hours in the NICU and those with defects of auricles, which prevented the performance of AABR, and infants born in another hospital of the city, who died or who have not completed all stages of the study.The sample was divided into two groups according to the date of birth of neonates and hearing screening procedures:Group I (GI): neonates born between March 2011 and March 2012 who underwent NHS in one stage;Group II (GII): neonates born between April 2012 and March 2013, who underwent two-stage NHS (test and retest).Initially, doctors of the NICU team selected newborns that could complete the hearing screening on the days when the screening was performed, considering the general conditions of the neonate and the probable date of hospital discharge. Then, identification data, birth conditions, and risk factors for hearing loss present in clinical history were collected from medical records and/or discharge summary of the child .Newborns were screened by an audiologist, using AABR  with click-type stimulus on the intensity of 35 dB NA, preferably before hospital discharge. When it was not possible, the doctor responsible for the discharge scheduled the screening.AABR was accomplished by placing electrodes, according to the equipment manual, with white on the top of the forehead, black on the cheek, and red on the nape of the neck. An ear probe tip was used in one ear at a time, randomly, according to the position of the neonate in the cradle or with the mother in natural sleep. The test result was recorded in the book of vaccination and/or the newborn medical record. Infant passed the NHS when responding to clicking the 35 dB bilaterally. In this case, the neonate was discharged or referred for follow-up of auditory and language development at 6 months when presenting risk factors associated with delayed-onset, or progressive hearing loss.In case of the referred result, the procedures were different for groups GI and GII.Group I: infants were directly referred for complete audiological evaluation.Group II: infants underwent retest with the same initial screening procedure (AABR), performed in the hospital, by appointment, approximately one month after discharge. Newborns who passed the retest were referred for follow-up of auditory and language development. Those who failed retest were referred for complete audiological evaluation.anamnesis with parents and otoscopic exam;assessment of the middle ear tympanometry and the ipsilateral acoustic reflex at 500 to 4000 Hz, with a 1000 Hz probe tone in infants aged 0 to 6 months and with a 226 Hz in infants older than 6 months, conducted through a Middle Ear Analyzer-Impedance Audiometer, AT235h, Interacoustics;transient evoked otoacoustic emission (TOAE) captured by 292 ILO USBII equipment;evoked auditory brainstem response (ABR), electrophysiological threshold and integrity of the auditory pathway conducted through Eclipse EP 25, Interacoustics with insert earphones. The skin was cleaned with alcohol and an abrasive paste before applying the conducting gel. Surface electrodes were the active electrode (Fz) and ground (Fpz) on the forehead, and the reference electrodes on the right (M2) and left (M1) mastoids. Impedance between electrodes was less than 3 KOhms, as recommended by the manufacturer. A nonvariable intensity of 80 dBHL was used to assess the integrity of auditory pathways from the inner ear to the brainstem and allowed the identification of possible changes in this path. The parameters analyzed were absolute latencies of waves I, III, and V; latency and interpeaks I-III, III-V, and I-V; wave V amplitude compared to the amplitude of wave I; interaural difference in interpeak I-V or latency of wave V. The electrophysiological threshold was obtained by the stimulus presented at decreasing intensities, until the lowest intensity that triggered the onset of wave V for click (2000 a 4000 Hz) and 500 and 1000 Hz tone bursts. Stimulation was repeated twice to check the reproducibility of track and ensure the presence of response.The audiological evaluation was performed at the Laboratory of Diagnostic Audiology Assessment of Children at Unicamp. The child remained in natural sleep and the procedures were as follows:It was considered normal hearing when the infant had lower electrophysiological thresholds/equal to 30 dBHL, absolute and interpeak within the expected values for gestational age latencies, and responses as expected in other procedures.Cases with abnormal results were referred to ENT assessment in the Clinical Hospital of Unicamp and the children were subjected to physical and/or imaging examination by an otolaryngologist physician (ENT) team.The child's hearing was classified as normal or hearing loss from the joint analysis of the outcome of audiological and ENT evaluation. The diagnosis of conductive hearing loss occurred when the otorhinolaryngological evaluation revealed disorders of the middle ear by otoscopy associated with increased values of waves I, III, and V absolute latencies and normal I-III, II-V, and I-V interpeaks, plus no transient or distortion product otoacoustic emissions, tympanogram type B or C, and no acoustic reflex. Sensorineural hearing loss was diagnosed when waves I, III, and V absolute latencies and I-III, III-V, and I-V interpeaks were normal or there was absence of wave I and prolongation of latencies of waves III and V or total absence of waves, depending on the degree of the hearing loss. Moreover, this diagnostic included electrophysiological thresholds greater than 30 dB, no TOAE, tympanometry type A, and presence or absence of acoustic reflexes, according to the degree of loss. The auditory neuropathy spectrum was characterized by absence of responses on ABR at the maximum intensity of the device (100 dB), presence of cochlear microphonic, presence of TOAE, tympanogram type A, and no ipsilateral acoustic reflexes.All data collected in the child's records, as well as the results obtained by the hearing screening and other assessments performed, were recorded in a computerized database. Tables of the results were constructed. To compare groups I and II regarding qualitative characteristics, Chi-square test or Fisher exact test was applied. The same tests were used to study the outcome of the hearing screening and audiological diagnosis for GI and GII. To assess the risk of hearing loss according to the presence of risk factors, the prevalence ratio and its confidence interval were calculated. The significance level was 5%; data in which statistically significant differences were found are highlighted in bold. The SAS software version 9.2 was used for this analysis.The study sample consisted of 929 live newborns, 52.5% male, 66.1% preterm births, and 42.7% underweight (weight between 1500 and 2500 g).P = 0.44), gestational age (P = 0.91), and birth weight (P = 0.85), which gave the group homogeneity.The GI consisted of 488 newborns (NB) and GII consisted of 441 NB. There were no statistical difference between GI and GII and the variables gender (In n = 763).P < 0.0001), retest (P < 0.0078), and the audiological diagnosis (P < 0.0431). Neonates with this indicator showed a greater number of failures in hearing screening test and retest, and also in the diagnosis of hearing loss compared to normal hearing and conductive loss compared to sensorineural.It was found that the risk factor related to craniofacial anomalies involving the ear and temporal bone significantly influenced the outcome of the NHS test (P = 0.0598). In this case, the diagnosis of normal hearing was more frequent when compared with the diagnosis of hearing loss (P = 0.0236). The statistical difference also occurred when comparing the type of hearing loss, with the most common being the conductive type (P = 0.0425).The risk factors for birth weight less than 1500 g, prematurity, neonatal and congenital infections, and anoxia were studied linked because they are frequent in newborns from NICU. It was verified that they did not affect the outcome of the NHS (P < 0.0001) and in GII in the retest (P = 0.0011).The results showed higher failure rate in neonates with genetic syndromes that usually express hearing loss, both in GI  presented normal hearing. Hearing loss occurred in 35.5% (16) of infants of which 20.0% (9 cases) were conductive, 13.6% (6 cases) sensorineural, and 2.2% (1 case) with auditory neuropathy spectrum disorder . In GI, In GII, normal hearing occurred in 12.5% of cases. The hearing loss occurred in 75% of children, of which 62.5% were conductive and 12.5% sensorineural. Comparing these results with the groups, it was found that there was a significant difference . In GII,The incidence of hearing loss in the sample was approximately 3%, of which 0.91% were sensorineural, 0.13% with auditory neuropathy spectrum disorder (ANSD), and 1.8% conductive . The higThe most common etiology is otitis media, clinical entity characterized by acute inflammation of the mucosal lining of the middle ear. It is the most frequent diagnosis in children and becomes less common with advancing age. Children presented 65% of risk of developing an episode during the first 24 months. Otitis media is considered a highly prevalent disease in childhood, with higher peak incidence between 6 and 24 months of age, and the second peak incidence between 4 and 7 years old. Inflammation of the middle ear cavity results from the interaction of several factors. The most important factors are infection and auditory tube dysfunction, which result from immaturity of the immune system and also the structural and functional immaturity of the auditory tube . AlthougThe cases of sensorineural hearing loss are in habilitation (hearing aids or cochlear implant) and speech rehabilitation process. Paludetti et al.  considerThe ANSD occurred in one neonate from GI, who was born at 38 weeks of gestational age and 3175 g of weight, remained 55 days in NICU, and presented the risk factors neonatal asphyxia and mechanical ventilation for prolonged periods, which were the probable etiology of the alteration. The infant refer NHS bilaterally on AABR to 45 dB and diagnosis showed no waves in the ABR, presence of cochlear microphonic and otoacoustic emissions, tympanometric curve type A, and no acoustic reflex. A study realized with temporal bones of preterm revealed 27% of selective loss of inner hair cells, higher prevalence than term infants (41% versus 28%) . The infThe results of NHS were analyzed, step test, considering the age in days after birth and postconceptual age at which screening was applied and the results passed or referred . It was None of the infant GI or GII with postconceptual age less than 34 weeks failed the NHS, so the postconceptual age did not influence the results of NHS. The literature recommended that NHS conducted through AABR be applied from 34 weeks so there is no influence of the maturity of the auditory system, especially up the brainstem, in the pass/fail result . The finChildren who passed, but presented risk indicators for progressive and/or late hearing loss, were referred for hearing and language follow-ups at 6, 12, 18, and 24 months. This procedure also aimed to detect any upward or conductive losses that, given the characteristics of procedure adopted, passed the NHS. The literature indicated that the follow-ups conducted in infants in the NICU showed that hearing deteriorated in a small percentage of children . It was The dropout at various stages of NHS and audiological diagnosis was computed. No significant difference was found between evasion in different stages for GI and GII. Considering the screening, stage test, and diagnostic in GI, dropout was 21.7%. In the retest performed in GII, dropout was 2% and, considering the whole process, about 24% . TherefoThe evasion of hearing care programs is a global reality. States with programs of early detection and intervention more developed in the United States include prevalence 2-3/1000 infants with hearing loss, but there are states with lower prevalence, probably due to dropout. In the United States, only 55.2% of children who failed the hearing screening attended diagnosis and 64.3% are in rehabilitation programs , 23. EvaAdherence to hearing health programs is a challenge that must be overcome in order to achieve the goals of early detection of hearing loss. The low attendance at prenatal consultations (one to three visits), having more than one child in the family, the absence of a partner, in addition to low maternal education, lack or low number of available services, and lack of knowledge of responsible membership interfered in the adhesion in child health program . SocioecThe most common risk factors in the studied sample were NICU patients who stayed for more than 5 days, preterm birth, use of mechanical ventilation, being small for gestational age, exposure to ototoxic drugs, weight of  less than 1500 g, and Apgar score of 0–4 in the first minute . These rIt was found that craniofacial anomalies involving the ear and temporal bone significantly influenced the outcome of the hearing screening and audiological diagnosis. Neonates with this indicator showed a greater number of failures in the hearing screening, diagnosis of hearing loss compared to normal hearing, and conductive loss in relation to sensorineural.We compared the results obtained in hearing screening and audiological diagnosis of neonates who had at least one indicator of the risk factors, including birth weight less than 1500 g, preterm birth, anoxia neonatal, and congenital infections with the results of neonates who presented the other studied risk factors. This analysis was conducted because they are often present in clinical histories of newborns that remained in the NICU. It has been found that these indicators did not influence the result of hearing screening. Comparing the diagnosis of normal hearing and hearing loss, there was a significant difference, with normal hearing the most frequent result. The statistical difference also occurred when comparing the type of hearing loss; the most frequent was conductive hearing loss.Genetic syndromes that usually express hearing loss significantly influenced the result of  NHS, step test for GI and GII and retest stage for GII, resulting in higher failure rate in neonates with this factor. GI and GII preterm neonates showed a lower percentage of referral than the others in the test stage. The risk indicator neonatal asphyxia influenced the diagnosis. The only neonate with this indicator showed sensorineural hearing loss.Comparing our results with the literature, it was verified that low birth weight, mechanical ventilation, and growth retardation were significantly associated with reference to NHS, unlike our findings . LiteratIt is essential to consider the risk factors present in the clinical history of newborns, not to select candidates for hearing screening, but to serve as a guide to the most appropriate management for each case. It is imperative that infants with risk indicators that influenced the results of the hearing screening and diagnosis of hearing loss attend the necessary steps of the hearing child health program.It was not possible to perform universal neonatal hearing screening in the sample. The postconceptual age did not influence the result of hearing screening; therefore, it should not be unnecessarily delayed. It should be performed as soon as possible, only considering the clinical condition of the infant.Retesting reduced the failure rate, the rate of false positives and did not interfere with dropout, so it is a recommendable stage in ICU hearing screening programs.The incidence of hearing loss was 2.9%, considering sensorineural hearing loss, conductive hearing loss, and auditory neuropathy spectrum. The incidence of sensorineural hearing loss was 1.04%.Strategies to minimize the evasion in child hearing health programs should be implemented.Infants with craniofacial anomalies involving the ear and temporal bone showed a higher number of failures in the hearing screening of hearing loss diagnosis compared to normal hearing and of conductive loss in relation to sensorineural. The risk factors of genetic syndromes that often express hearing loss and prematurity had significant influence on the outcome of hearing screening.From the analysis of the results the following was concluded.


Some of the most extreme marine habitats known are the Mediterranean deep hypersaline anoxic basins . Brines of DHABs are nearly saturated with salt, leading many to suspect they are uninhabitable for eukaryotes. While diverse bacterial and protistan communities are reported from some DHAB water-column haloclines and brines, the existence and activity of benthic DHAB protists have rarely been explored. Here, we report findings regarding protists and fungi recovered from sediments of three DHAB  haloclines, and compare these to communities from sediments underlying normoxic waters of typical Mediterranean salinity. Halocline sediments, where the redoxcline impinges the seafloor, were studied from all three DHABs. Microscopic cell counts suggested that halocline sediments supported denser protist populations than those in adjacent control sediments. Pyrosequencing analysis based on ribosomal RNA detected eukaryotic ribotypes in the halocline sediments from each of the three DHABs, most of which were fungi. Sequences affiliated with Ustilaginomycotina Basidiomycota were the most abundant eukaryotic signatures detected. Benthic communities in these DHABs appeared to differ, as expected, due to differing brine chemistries. Microscopy indicated that only a low proportion of protists appeared to bear associated putative symbionts. In a considerable number of cases, when prokaryotes were associated with a protist, DAPI staining did not reveal presence of any nuclei, suggesting that at least some protists were carcasses inhabited by prokaryotic scavengers. Redox boundaries in marine sediments can have significant geochemical gradients, transitioning from fully aerated to anoxic (lack of detectable dissolved oxygen) conditions within short vertical distances e.g., . These cMicrobial eukaryotes inhabiting marine chemoclines can be numerous compared to those from nearby more aerated sites e.g., . AdditioFigure 1 and also see Figure 1 in Table 1). Further details on the brine chemistries are available in The deep bathyal Mediterranean has numerous deep hypersaline anoxic basins .These DHABs have been the topic of attention over the last decade. In particular, the water columns overlying these bathymetric features have received considerable study, for both prokaryote communities  and eukaThe goal of this study was to investigate the microbial eukaryotes in the redoxcline of Mediterranean DHAB sediments. Here we report results describing the eukaryotic community composition and abundances. We include results from classic morphologic analyses as well as from RNA-based sequencing efforts and a fluorescence-labeling approach to identify active eukaryotes. For comparison, we include data on microbial eukaryote communities obtained from adjacent normoxic sediments.Jason from 24 November to 6 December 2011. Most sediments for this study were collected with the typical Deep Submergence Lab (http://www.whoi.edu/groups/DSL/) Alvin-type pushcores  configured with a seal to prevent contamination during ascent. Cores were collected from nearby (within 100 m) aerated sediments, just upslope from each DHAB. Thus, control samples at Urania and Discovery were consistently from 1 to 3 m shallower water depths than the halocline samples. Because ROV Jason is neutrally buoyant, it was not possible to core deep in the DHAB brine. Thus, cores of deep halocline sediments were obtained by Jason reaching toward the brine with the ROV manipulator. The depth differential between halocline samples did not exceed 2 m. Given the waters depths for all samples were >3400 m, these depth differences are negligible.Samples were collected using the ROV Jason recovery, all cores were taken as quickly as possible (within 5 min) to the RV Atlantis environmental room set at 9.5°C (±0.5°C), until further processing. Cores for microelectrode profiling were placed in an N2-flushed glove bag and profiled as described below. Cores for quantification (counts) and imaging were first sampled for their bottom-water salinity, measured via hand-held refractometer. Many samples needed to be diluted up to 10-fold to register on the refractometer. The hand-held refractometer was not expected to provide precise salinity data, especially for the very high MgCl2 brine of Discovery, but was intended as a relative indicator between samples from different halocline horizons in any given DHAB. After salinity was recorded, each of these cores was sectioned into 1-cm intervals to a depth of 3 cm. Each sediment slice was preserved in either 3.8% formaldehyde or 3% TEM-grade glutaraldehyde in a 0.1 cacodylic acid sodium salt buffer. In some cases, there was no overlying bottom water due to full penetration or over-penetration of the corer. In these cases, salinity data are not reported. In instances where no salinity data was obtained from any given core, we estimated salinity using published literature (Table 1) and confirmed by our own water column high-range CTD data , extrapolated for specific core location relative to others with known salinity and the halocline boundary. We designate halocline samples by their relative location  in the following descending order: upper halocline, mid-halocline, lower halocline.Upon ROV in situ using the viability indicator CellTrackerTM Green CMFDA . CellTracker Green relies on active esterases, which are known to function in at least one DHAB brine (Jason dive (2–3 days later), these cores were removed from the seafloor per standard procedure and brought to the sea surface. Because only one dive was executed in L’ Atalante, those incubations were begun early on the dive and terminated after recovery later that day, resulting in incubation times of approximately 12.5 h. The injector cores were sectioned and preserved in glutaraldehyde as noted above.To avoid the possibility that pressure and temperature changes during ascent may cause death of some fraction of the eukaryotic community, a subset of cores were incubated AB brine . For theAB brine . During Atlantis, designated cores containing overlying site water were profiled for dissolved oxygen with a vertical resolution of 0.25 mm in a N2-filled glove bag using polarographic microelectrodes . Major modifications included introduction of three cycles of freeze-thaw , bead beating with 2 × 5 min. intervals on a horizontal vortexer, an overnight nucleic acid precipitation, and a 1-hour centrifugation during the precipitation step. In addition, we introduced two DNAase treatments using TurboDNAase . Removal of DNA was confirmed by PCR using general eukaryotic primers. RNA was purified using the MEGAclear kit . Reverse transcription of the purified RNA samples was performed using the QuantiTect kit . Tag-pyrosequencing of the eukaryotic small subunit rRNA (18S rRNA) gene was performed using PCR amplification of the V4 region of the 18S rRNA gene and the primer pair TAReuk454FWD1 (5′-CCAGCA(G/C)C(C/T)GCGGTAATTCC-3′) and TAReukREV3 (5′-ACTTTCGTTCTTGAT(C/T)(A/G)A-3′). In brief, a one-step, 30-cycle PCR reaction was performed using GoTaq polymerase . PCR conditions included: 94°C for 3 min, followed by 30 cycles of 94°C for 30 s; 55°C for 40 s; 72°C for 1 min; and a final elongation step at 72°C for 5 min. Following PCR, all amplicon products (ca. 450 bp) from different samples were purified using the MinElute Reaction Cleanup kit . In parallel, blank extractions and amplification reactions were performed to check for contamination. Distinct tags (multiplex identifiers) were used for each of the samples. Amplicon libraries were sequenced utilizing the Roche 454 FLX Titanium platform and reagents following manufacturer’s guidelines at the MR DNA  sequencing facility.Denoising of the flowgrams was performed using Acacia . ProcessAt WHOI, pushcore samples preserved in either glutaraldehyde or formalin on the ship were used for morphologic documentation and/or quantification. Percoll density gradients were used to isolate the eukaryotes , subjectBecause the filter impedes imaging, the specimen-laden side of a subset of filters from selected samples was blotted onto a drop of mounting media atop a microscope slide, covered with a coverslip and then sealed. The mounted slide was scanned for eukaryotes appropriate for imaging . Images were taken either with an Olympus BX51 upright epifluorescence microscope with DIC and phase contrast optics and an Olympus DP70 color digital camera or the Zeiss Axio Imager M2 and a Canon EOS Rebel color digital camera.Non-quantitative aliquots of samples incubated on the seafloor with CellTracker Green were similarly subjected to Percoll extraction, DAPI staining and/or imaging, although specimens were also examined under 480-nm excitation and 520-nm emission to determine if they had active esterases at the time of incubation.Figure 3A). In all but one of the halocline cores, oxygen was detectable in overlying waters . At the sediment-water interface of halocline cores, [O2] was typically below 21 μM. Oxygen remained detectable to at least 2.0 cm depth in 7 of the 12 halocline cores. In some halocline cores , oxygen remained detectable at the deepest measured horizon (2.5 cm). Oxygen was near the detection limit (<3 μM) throughout the top 2.5 cm in core 608 c 11 (Urania); this core was not analyzed for eukaryotes.Dissolved oxygen in control cores remained detectable to depths deeper than 1 cm (Table 2); the highest salinity measured in Discovery was 146. We reiterate that these salinities do not represent brine characteristics per se but allow relative comparisons between pushcores collected within each halocline.Salinity in bottom waters within 10 cm of the sediment–water interface of cores from Discovery and L’ Atalante varied widely. Salinity was not measured from cores collected in and near Urania DHAB. Control cores had bottom-water salinities of 41–50. The highest salinity measured was 220 . A number of morphotypes were observed . For example, what appeared to be a thecate foraminifera was observed in a control sample , but none of these were noted in halocline samples. Small (3–5 micron) eukaryotes that were seen under fluorescence microscopy appeared to be consistent with flagellates, dominated microscopic counts in terms of abundance, but the most photogenic protists were ciliates . Not all protists had discernable nuclei. When no nuclei or few prokaryotes via DAPI were observed, the specimen was deemed dead . In some cases, nuclei were visible in both DIC and DAPI images . In other cases, even though prokaryotic associates (see below) were discernable by DAPI staining, nuclei that should also be discernable via DAPI staining were not obvious . Some samples  had numerous filamentous bodies that were likely fungal hyphae or, perhaps, filamentous bacteria similar to Beggiatoa, although well-developed Beggiatoa mats were not observed. Moreover, Beggiatoa sequences were not detected in our samples . Metazoa that were found  are not considered in this contribution.Protist morphotypes were detected from every habitat  in each sample examined for this purpose , while others did not . Ectobionts were noted on a few specimens , and clumps of endobionts were seen in a halocline ciliate from L’ Atalante .Although protist populations were not quantified for presence of prokaryotic associates , many ciliates had endobionts . Such specimens were observed in normoxic and some halocline samples .In the aliquots examined for both CellTracker Green labeling and DAPI labeling, esterase-positive  protists were observed that had easily distinguishable nuclei, either in addition to or exclusive from any associated ecto- and/or endobionts . The mean protist density of the five halocline samples  was significantly higher than the protist density at the normoxic (Urania control) sample, .Densities of protists varied within haloclines at Discovery Basin and between basins  in the Discovery upper halocline, followed by both L’ Atalante halocline samples , and Urania halocline (82 reads) and Urania normoxic control (75 reads). For two of the samples analyzed, the number of curated reads was too low and these were excluded from the graphical representation. Discovery mid-halocline had a total of 8 reads, where most were Ustilaginomycotina Basidiomycota (not shown). Discovery lower halocline had a total of 6 reads, where most were Chlorophyceae (not shown).Figure 10). In four of these five samples, the majority of eukaryotic reads were fungal, with most being affiliated to Ustilaginomycotina Basidiomycota. L’ Atalante upper halocline was dominated by Chlorophyceae. Other groups with high diversity were Syndiniophyceae  and Dinophyceae  Dinophyta. Rhizarians were also present, sometimes in considerable proportions , mostly as Radiolaria.A higher number of orders of eukaryotic taxa based on SSU rRNA signatures was detected in the Urania aerated control sample compared to the halocline samples, implying that alpha diversity may be lower inside these DHABs. The only exception was L’ Atalante upper halocline, which also had relatively high diversity , protist numbers can rival those in near-surface waters, ∼1071 ± 117cells mL-1  samples. Eukaryote densities in the control (normoxic) sediments were roughly similar to densities of other protist communities in some oxygenated bathyal sediments  with published data on the seawater chemistry of each DHAB (Table 1). In general, higher protist densities occurred with higher concentrations of HS-, Mg2+, and SO42 to a threshold . Protist densities decreased with the highest concentrations of sulfide and magnesium. No relationship was apparent between salinity and protist density. Refractometer readings of salinity for athalassohaline brines such as Discovery are problematic, and therefore comparisons to thalassohaline brines such as Urania and L’ Atalante are difficult. Protist density may relate to the differing cation chemistries in each of the DHABs (Table 1) and whether encountered specimens were living or dead (see below). Statistical analyses to test for trends were not performed as protist data from replicate cores at each habitat and halocline site were not available.To determine whether protist density showed a relationship with salinity or chemical constituent, we plotted our protist density data . This pattern is somewhat similar to observations of another ciliate with multiple endobionts in a structured configuration, inhabiting chemocline sediments  yet presence of copious endo- or ectobionts evidenced by DAPI staining. Prokaryote presence within degrading foraminifera cytoplasm has been documented in hydrocarbon seep habitats , at least for some constituents. More specifically, for Mg2+ and HS-, it is plausible that the observed protist peak abundances coincide with the approximate maximum tolerance of protists for the constituent being considered . Regarding Mg2+, although an upper limit for prokaryotic life may be ∼2 M . Similarly, DHAB environments exceeding ∼1.5 mM HS-  and ∼200 mM SO42-  may not support living protist populations. Thresholds probably also depend on concentrations of additional constituents and their possible synergistic interactions in these brines, so generalizations involving habitats with typical marine salinities are likely invalid.The presence of pickled protists may explain some of the observed trends of protist abundance (densities) with respect to water chemistry . Importantly, we do not assert positive DAPI staining indicates viability, as we are aware that DNA can be preserved in DHAB brines  in the anoxic lower halocline water layer of another DHAB, Thetis  leads to a decrease in relative abundance of selected groups, e.g., a decrease in abundance within Dinophyceae, Cryptophyceae, Centroheliozoa, and Acantharea in the lower halocline sample from L’ Atalante basin relative to the upper halocline. The dominance of chlorophyte signatures in the Urania basin upper halocline sample suggests decreased protist diversity possibly due to the relatively high sulfide concentrations there.The application of metatranscriptomics to these communities may be the only feasible means at present to fully resolve eukaryote activity  and their inhabitation of these DHAB haloclines . Future efforts may reveal novel taxa and heretofore unknown biochemical pathways in these spatially restricted benthic habitats.Joan M. Bernhard and Virginia P. Edgcomb designed this study and drafted the manuscript. Joan M. Bernhard, Konstantinos Kormas, Maria G. Pachiadaki, and Colin Morrison collected the samples. All authors performed analyses and contributed to data analysis. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Sir,OPA1-related auditoryneuropathy: site of lesion and outcome of cochlear implantation’ published by et al. (2015)were the first to show that it is an important cause of hearing impairment in a geneticallydefined subset of these patients.We read with interest the article entitled ‘et al. (2015) comprehensively investigated the auditory phenotypein subjects with both missense (n = 10) and haploinsufficiency(n = 11) mutations in OPA1, which were first described inpatients with autosomal dominant optic atrophy . Here, a click stimuluswith alternating polarity was delivered at a suitable sensation level as to give a clearresponse. The sensation level was predetermined by the mean hearing level by ear at 2/4 kHz:≤40 dB HL used click stimulus at 70 dB nHL, 40–60 dB HL used click stimulus at 80 dB nHL,>60 dB HL used click stimulus at 90 dB nHL. Contralateral masking was applied whenrequired.On average, 2000 responses were obtained on each side and these were bandpass filteredbetween 8 and 15 Hz. Waveforms and latencies of waves I–V were calculated and compared againstage-matched values. Where the I–V interval was not available wave III–V and absolute V latencywere calculated. Cochlear function was specifically tested using transient evoked otoacousticemissions (TEOAEs) (click stimulus 84 dB SPL) for outer hair cell function and psychoacoustictuning curves (PTCs) for both outer and inner hair cell function. Measurement of PTCs is thegold standard test for the detection of cochlear dead regions . These rUsing the ‘SWPTC’ programme, PTCs were measured using signal frequencies of 1, 2 and 4 kHz. The foun = 11) presented with a rangeof hearing levels: normal (n = 3/11); mild-to-moderate high-frequency loss; andsevere-to-profound loss at all measured frequencies . Patients with pathological mtDNA mutations oftenhave a mixture of mutated and wild-type mtDNA molecules (heteroplasmy). Although there was apositive correlation between mean age-corrected PTA and urinary heteroplasmy levels (range25–66%), this did not reach statistical significance . No relationship was found between hearing levels and bloodheteroplasmy levels (range 3–30%).Patients with the m.3243A > G mutation , III-V  and I-V  were within the normal range for the remaining patients  A and B. PTC analysis was performed for 9/11 patients. Two patients were unable to complete the testdue to the severity of their hearing impairment. For patients with normal or mild hearingloss, no tip shifts were detected . For patients withmoderate-to-severe hearing loss, two patients showed tip shifts at 1 kHz consistent withdead regions (tip shift ratios 0.79 and 1.11) A. POLG patients studied . Moderate high-frequency hearingloss was present in two patients and one patient had a moderate loss at all measuredfrequencies. One patient showed a predominantly moderate mid-frequency loss  either had normal hearing (n =5/10) or a moderate high-frequency hearing loss . TEOAEs were present for 9/10 patients. The interpeaklatencies of ABR waves I-III , III-V  and I-V  were within the normal range forall patients. PTC analysis was performed for 9/10 patients. One patient was unable toperform the test due to ataxia. No dead regions were detected . See The SPG7 patients (OPA1 mutations harbouringhaploinsufficiency (n = 4) or missense (n = 1)OPA1 mutations. Patients carrying haploinsufficiency mutations had eithernormal hearing (n = 3) or a mild high-frequency loss . TEOAEs were absent for one patient withhigh-frequency loss. One patient with a missense OPA1 mutation had amoderate high-frequency loss  with absentTEOAEs. The interpeak latencies of ABR waves I-III , III-V  and I-V  were in the normal range for allpatients. PTC analysis was performed for 5/5 patients and did not reveal any dead regions.We also included a cohort of patients with POLG disease and similar to m.3243A > G, this seems to be cochlear inorigin. Likewise, mild cochlear hearing impairment or normal hearing was found in ourSPG7 and OPA1 patient cohort.In agreement with previous studies, we found hearing levels for patients carrying them.3243A > G mutation in the range from normal to profound loss, with absent TEOAEs butpreserved ABRs, indicating that the hearing loss is cochlear in origin , we found no evidence of isolatedauditory nerve impairment in our patient cohort.In conclusion, our data suggest that both mtDNA mutations and a range of nuclear-geneticmitochondrial disorders cause hearing loss through cochlear dysfunction. The presence ofdead regions in a small number of patients carrying the m.3243A > G mutation suggeststhat mtDNA point mutations cause specific dysfunction of inner hair cells. On the otherhand, the absence of cochlear dead regions with loss of TEOAEs, but with normal ABRs,suggest that the hearing loss in patients with SPG7and POLG mutations, who have not been systematically studied before.Further improvements in understanding the pathogenesis and natural history of thesedisorders are important to provide better prognostic information to patients and to informfuture trial design.Hearing loss has a negative impact on quality of life and it is often overlooked inpatients with mitochondrial disease who can present with a range of complex heterogeneoussymptoms. Our data confirm that sensorineural hearing loss is an important pathologicalfeature in a range of mitochondrial diseases, including patients with POLG mutations.P.F.C. is a Wellcome Trust Senior Fellow in Clinical Science (101876/Z/13/Z), and a UK NIHRSenior Investigator, who receives support from the Medical Research Council MitochondrialBiology Unit (MC_UP_1501/2), the Wellcome Trust Centre for Mitochondrial Research(096919Z/11/Z), the Medical Research Council (UK) Centre for Translational Muscle Diseaseresearch (G0601943), EU FP7 TIRCON, and the National Institute for Health Research (NIHR)Biomedical Research Centre based at Cambridge University Hospitals NHS Foundation Trust andthe University of Cambridge. P.J.K. is a Wellcome Trust Clinical research fellow(101700/Z/13/Z). P.Y.W.M. is supported by a Clinician Scientist Fellowship Award (G1002570)from the Medical Research Council (UK), and also receives funding from Fight for Sight (UK)and the UK National Institute of Health Research (NIHR) as part of the Rare DiseasesTranslational Research Collaboration. The views expressed are those of the author(s) and notnecessarily those of the NHS, the NIHR or the Department of Health. We acknowledge MedicalResearch Council (MRC) Centre Mitochondrial Disease Patient Cohort: A Natural History Studyand Patient Registry (REC ref number:13/NE/0326) for patient screening and data provision.We are grateful to Alex Bright, Research Nurse, for her work recruiting individuals with them.3243A>G and


The aims of the present study were to investigate the ability of hearing-impaired (HI) individuals with different binaural hearing conditions to discriminate spatial auditory-sources at the midline and lateral positions, and to explore the possible central processing mechanisms by measuring the minimal audible angle (MAA) and mismatch negativity (MMN) response. To measure MAA at the left/right 0°, 45° and 90° positions, 12 normal-hearing (NH) participants and 36 patients with sensorineural hearing loss, which included 12 patients with symmetrical hearing loss (SHL) and 24 patients with asymmetrical hearing loss (AHL) [12 with unilateral hearing loss on the left (UHLL) and 12 with unilateral hearing loss on the right (UHLR)] were recruited. In addition, 128-electrode electroencephalography was used to record the MMN response in a separate group of 60 patients  and 20 NH participants. The results showed MAA thresholds of the NH participants to be significantly lower than the HI participants. Also, a significantly smaller MAA threshold was obtained at the midline position than at the lateral position in both NH and SHL groups. However, in the AHL group, MAA threshold for the 90° position on the affected side was significantly smaller than the MMA thresholds obtained at other positions. Significantly reduced amplitudes and prolonged latencies of the MMN were found in the HI groups compared to the NH group. In addition, contralateral activation was found in the UHL group for sounds emanating from the 90° position on the affected side and in the NH group. These findings suggest that the abilities of spatial discrimination at the midline and lateral positions vary significantly in different hearing conditions. A reduced MMN amplitude and prolonged latency together with bilaterally symmetrical cortical activations over the auditory hemispheres indicate possible cortical compensatory changes associated with poor behavioral spatial discrimination in individuals with HI. Spatial hearing refers to the ability of the human auditory system to identify, discriminate, localize and communicate a source of sound in a complex environment . It is gA number of studies have indicated that the ability to detect a fine difference in binaural information is affected by sensorineural hearing loss, which causes deficit in the perception and analysis of the frequency and temporal information in sound inputs ,7,8. ForAlthough several studies have reported that ability of spatial hearing is affected by hearing impairment (HI), due to the poor binaural hearing effect ,12,13,14Spatial discrimination is closely associated with extracting binaural sound information from the auditory system, which helps listeners to identify target and understand speech, particularly in difficult listening situation . SpatialThere are however few studies that have investigated the influence of binaural hearing on spatial discrimination using the MAA in profound hearing impaired people using hearing assistant devices ,22,23,24To investigate the effect of source angle on spatial discrimination, MAA has been measured and compared in bilateral CI participants and age matched NH controls using white noise bursts at frontal and lateral position. SignifiIt is noteworthy that binaural hearing effects on spatial discrimination described above were obtained from profound hearing impaired individuals with bilateral fitted CIs, rather than natural hearing impaired conditions with various audiological configurations  as well as the degree of hearing losses. The influences of these factors remain unclear.Much evidence shows that central auditory processing is essential for accurate discrimination of sound in NH individuals ,26. MismA study by Richter et al.  found coPrevious studies also suggest that cortical compensatory plasticity may be correlated with speech recognition ability . CampbelIn the present study, the aims were to investigate acoustic spatial discrimination ability at the midline and lateral positions and to explore the possible central processing mechanism in HI individuals with different binaural hearing conditions using the (MAA) and MMN responses respectively.Informed consent was obtained from all participants in compliance with a protocol approved by the Institution Review Board of The Sun Yat-sen Memorial Hospital at Sun Yat-sen University of China.12 NH participants and 36 participants with sensorineural hearing loss (Group A), including 12 participants with SHL and 24 participants with AHL [12 with unilateral hearing loss on the left (UHLL) and 12 with unilateral hearing loss on the right (UHLR)] took part in the MAA task. A second group (Group B) was recruited for the MMN test. Group B comprised 20 NH and 60 participants with sensorineural hearing loss, which included 20 UHLL, 20 UHLR and 20 SHL participants.Participants ranged in age from 18 to 60 years old. All were recruited for both experiments via ENT/Audiology clinics. The general information of the participant is summarized in The inclusion criterion for the SHL group was an average between-ear difference in hearing level of less than 15 dB at 0.5, 1, 2 and 4 kHz. In contrast, the AHL group had an average between-ear difference in hearing level of greater than 15 dB  and group , nor did the interaction between hearing condition and group . Statistical analysis for the hearing thresholds of the better  and worse  ears revealed no significant differences between the two experimental groups.A Chi-squared test revealed no significant difference in gender ratios  = 29.78, p<0.001], but not direction . Further Bonferroni-adjusted comparisons for different hearing conditions revealed that the averaged MAA was significantly smaller in the NH (4.88°) group than in the SHL (10.26°), UHLL (13.69°) and UHLR (10.73°) groups. In addition, the averaged MAA was significantly smaller in the SHL group than the UHLL group . A significant interaction was found among the factors of hearing condition, angle and direction . When the interaction was further broken down by the hearing condition, follow-up analyses showed that the MAA were significantly smaller for signals originating from the 0° position than those originating from the 45° and 90° positions in the NH group   = 0.151, p = 0.705] groups.Values of MAA performance were subjected to a 4  x 3  x 2  repeated-measures ANOVA. There were significant main effects of hearing conditions  . The samp<0.001] . Howeverp<0.001], which led to further analysis, revealing that the MAA threshold for the 90° position was significantly smaller than the thresholds obtained from the 0 and 45° positions when the sound was presented from the left side . However, a significant smaller MAA threshold was found at the 0° position than at 90° positions on the right side   = 38.26, p<0.001]. Moreover, a significant interaction was also found between angle and direction  .p = 0.001]. A significant interaction was also found between the angle and direction , and the follower-up analyses showed that the MAA threshold for the 90° position was significantly smaller than the thresholds obtained from the 0 and 45° positions when the sound was presented from the right side . In addition, a significant smaller MAA threshold was found at the 0° position than at 45° and 90° on the left side   = 17.93, p<0.001] .t-test. All MMN amplitudes showed significant statistical differences. As shown in As mentioned earlier, the MMN components were verified using a one-tailed p = 0.002)] and angle . Participants had larger MMN amplitudes at sound signals presented from 90° (-1.512 uV) than from 45° (-1.167uV). Further Bonferroni-adjusted comparisons for different hearing conditions revealed significantly greater amplitudes in the NH (-1.897 uV) group than those obtained from the UHLL (-1.179 uV), UHLR (-1.212 uV) and SHL (-1.068 uV) groups.p = 0.008], showing that NH participants had shorter latency (182.63 ms) than participants with UHLL (211.74 ms) and UHLR (217.30 ms). However, the main effect for angle and direction did not reach significant level For MMN latency analysis, there was a significant effect of hearing condition , hemisphere  and angle . A significant interaction was found between hearing and hemisphere , as well as between hemisphere and direction . In addition to explore the cortical plasticity of spatial auditory processing in individuals with different binaural hearing conditions, interaction was further broken down by hearing condition.To investigate central processing in the left and right cortical areas, the average of the signals received by electrodes F2, F4 and Fc6 and the average of the signals received by electrodes F1, F3, and Fc5 were chosen for statistical analysis of the activity in the left hemisphere and right hemisphere, respectively. The reason for choosing these electrode sites was that the MMN responses to spatial deviant positions were primarily evoked at these frontal-central electrodes , which wp<0.001]. Follow-up analyses showed that the right hemisphere was more highly activated by a deviant sound emanating from the left side than that from the right side . In contrast, a larger response over the left hemisphere was observed when the participants perceived sounds from the right side instead of the left side   = 25.85, = 0.011] .p = 0.027]. However, no significant difference in the MMN responses of the hemispheres was found when the sounds emanated from the right side   = 4.95, p = 0.043] for the SHL group. Further analysis showed the MMN response was significantly larger in the right hemisphere than in the left hemisphere when the SHL participants discriminated sounds emanating from the left side  .p = 0.011] and significant interaction between hemisphere and direction . To investigate the hemisphere effect on different direction, further RM-ANOVA was carried out with hemisphere and angle. This analysis showed significant main effects in angle  as well as interaction between hemisphere and angle  for left direction. A significantly larger MMN amplitude was found in the right hemisphere compared to the left hemisphere when UHLL participants received deviant sounds from 90° on the left direction . In contrast, no significant hemisphere effect was found when the sounds were presented from the right direction .For the UHLL group , there wp = 0.004]  = 12.826, p = 0.003] only for right direction. Sounds originating from the 90° position on the right direction induced a significantly larger MMN response in the left hemisphere than in the right hemisphere . On the contrary, no significant hemisphere effect  or interaction  were found for left direction.A significant interaction between hemisphere and direction was found for the MMN amplitude in the UHLR group  . SimilarIn this study, behavioral MAA and electrophysiological MMN techniques were used to examine auditory spatial discrimination and related cortical compensatory plasticity associated with conditions of hearing impairment. Overall MAA results showed that spatial discrimination was reduced with hearing impairment, although listeners with SHL had preserved the ability to spatially discriminate at a higher resolution than listeners with AHL and also exhibited a discrimination pattern similar to that of the NH subjects. In addition to the reduction in spatial discrimination an alteration in the spatial discrimination pattern was found in the listeners with AHL. Furthermore, significantly reduced MMN amplitude and prolonged latency as well as significant alteration in cortical activation was found in HI people.It is generally accepted that MAA measurement is a useful tool for examining auditory spatial discrimination ,19,20. IIn the hearing impaired (HI), although people with SHL showed a discrimination pattern similar to that of NH subjects, their auditory spatial-discrimination ability was generally reduced. This implies that the individuals with symmetrical binaural hearing made good use of binaural cues, but were less efficient due to the poor hearing sensitivity and possible frequency resolution .The present study also reveals that impairment of the peripheral auditory pathway also exerts a negative effect on spatial hearing discrimination, although the processing and encoding of interaural time and intensity difference information was mainly extracted in the central auditory system ,49. One Compared to subjects with SHL, listeners with AHL exhibited greater deficits in their spatial-discrimination ability, limited degree of binaural processing and greater variability in their responses in the MAA test. It is well established that listeners with SHL outperform listeners with AHL in spatial localization and sound-source segregation in complex environments by taking advantage of their binaural hearing ,6,11. ThIn addition to the significant reduction in spatial-discrimination ability, an interesting finding is the alteration in spatial discrimination pattern in listeners with AHL. Listeners with AHL demonstrated better spatial resolution at 90° on the affected side than at any other position (such as the midline position). This phenomenon may be attributed to shifting the spatial position of zero IID  from the midline in normal hearing status to a specific lateral side (for hearing impairment) . EvidencThe results obtained from the present study showed increased MMN latency and decreased amplitude in HI individuals, consistent with previous findings ,52. For For people with NH, the MMN responses indicate that auditory stimuli can strongly activate the contralateral auditory cortex, which is supported by related neurophysiological theory on the auditory input to one ear being projected to the contralateral auditory cortex ,57,58. HIndeed, established and more recent studies have advocated a form of cortical compensatory plasticity by demonstrating increased activation of the temporal auditory cortex responding to visual stimuli in association with poor speech/auditory outcomes due to hearing impairment ,37,38,39Furthermore, a recent MMN study by Bottari et al.  demonstrIn the present study, the finding of increased ipsilateral and reduced contralateral temporal activation in responding to lateral spatial stimuli may suggest the occurrence of possible cortical compensatory changes during discrimination of auditory spatial stimuli in parallel with increased temporal activation to visual stimuli, even in mild to moderately severe HL individual. Therefore, taken together, poor behavioral spatial discriminating outcomes, together with the evidence of decreased in MMN amplitude and prolonged latency, as well as the increased activation in bilateral symmetrical temporal areas during passive listening in various HL groups, indicates degraded behavioral spatial discrimination and possible cortical compensatory plasticity in people with HI.Assessment of cortical compensatory plasticity due to hearing loss using either objective or behavioral measurements will become increasing relevant in the clinical setting in terms of selecting hearing-assistance strategies for HI people . In addition, it would be helpful to explore whether auditory training  or other rehabilitative procedures in conjunction with hearing aid rehabilitation may alleviate the cortical re-allocation or re-training of auditory cortices in HI individuals and consequently re-activate normal cortical neural networks during auditory processing.A potential limitation of the present study was an inability to conduct a correlation analysis between the capabilities for spatial discrimination and the MMN responses due to the complexity and duration of the experiment, which could cause poor compliance and high drop out of participants. According to the overall aims of the present study, even though the participants for the MAA and MMN experiments were recruited from these two separate groups, the present study design and collected data were appropriate and valid because they shared similar characteristic for genders, age distribution and hearing threshold. Future research is needed to obtain evidence of a correlation between cortical compensatory plasticity and reduced spatial discrimination ability after hearing impairment. Moreover, it would be interesting to explore spatial discrimination ability of HI people in the complex environments (such as the presence of background noise).Spatial discrimination ability at midline and lateral positions vary significantly in different hearing conditions. The MAA thresholds obtained from the NH participants are significantly better than those in the HI participants. Moreover, the smallest MAA threshold was obtained at the midline position in NH participants and SHL participants However, for listeners with AHL, the best spatial resolution in terms of the MAA thresholds was found at the 90° position of the affected side. Furthermore, a reduced MMN amplitude and prolonged MMN latency together with bilaterally symmetrical cortical activation over the auditory hemispheres indicate possible cortical compensatory changes associated with poor behavioral spatial discrimination in individuals with HI.


According to World Health Organization (WHO) 2001 statistics, hearing disorders are the most common congenital disease, and the incidence rate among high-risk newborns is as much as ten times as high as that in healthy neonates. However, 78% of screening test failures are well-baby nursery babies. The Joint Committee on Infants’ Hearing (JCIH) has emphasized the importance of early diagnosis and treatment in neonates with hearing impairments in order to preserve their maximum linguistic skills. The aim of our study was to compare the prevalence of hearing loss among babies in the neonatal intensive care unit (NICU) and the rooming-in unit (RIU).Neonates born in three hospitals in Mashhad between 2008 to 2010 were studied prospectively and screened for auditory disorders using the oto acoustic emission (OAE) test at the time of discharge and 3 weeks later. To confirm hearing loss, the auditory steady state response (ASSR) test was used among those participants who failed both OAE tests.Two-thousand and sixty-three neonates from the NICU were screened and compared with a control group consisting of 8,724 neonates from the RIU or the well-baby nursery. At the end of the study, hearing impairment as confirmed by failure in the ASSR test was diagnosed in 31 neonates (26 in the control group [0.30%] and five in the NICU group [1.94%]).In our study, the prevalence of hearing disorders among NICU neonates was 6.5-times greater than that among babies from the RIU or well-baby unit. This observation demonstrates the importance of universal screening programs particularly for high-risk population neonates. According to World Health Organization (WHO) statistics, nearly 250 million persons were affected by hearing impairment of some description in 2001, with auditory disorders being the most common congenital disease . HoweverDifferent studies have reported various risk factors for hearing loss ,12. HoweThe aim of our study was to compare the prevalence of hearing loss in neonatal intensive care unit babies (high-risk group) with well-baby nursery babies (control group) using OAE and ASSR tests.This prospective study was conducted from November 2008 to November 2010 in three university-related hospitals in Mashhad, Northeast Iran. Neonates born in Imam Reza, Qaem or Ommol Banin Hospitals were screened for auditory disorders in order to evaluate hearing impairments.All neonates before discharge were screened, whether they were in the neonatal intensive care unit (NICU) (case group) or in the well-baby rooming-in unit (RIU) (control group). The OAE test  was used as the first step in the screening program. Neonates who failed in the primary test were followed-up with a second OAE test after a 3-week gap. Failure in the secondary test led to hearing loss being confirmed in the ASSR test . The frequency of hearing impairment was assessed, and data analysis was performed using SPSS software.A total of 263 neonates from the NICU participated in this study (case group). Overall, 185 cases (70.3%) failed in first OAE test, of whom 180 neonates (97.3%) participated in the secondary OAE test to confirm their first test results. One hundred cases  were referred again after the second OAE test. Ninety-nine neonates  underwent the ASSR test. Unilateral or bilateral hearing impairment was confirmed in five neonates who failed the 25 db in four 500, 1000, 2000,4000 hearing thresholds in ASSR test. In other words, 1.9% of 263 NICU participants had a hearing impairment in the case group.In the control group 8,724 well newborns from the RIU participated; of whom 1,046 cases failed in the first OAE test (12.0%). Eight-hundred and twenty-six neonates (79.0% of those who failed the first OAE test) participated in a second OAE test, which was failed by 419 neonates  -17. In o±3%) . In a 20±3%) . It is k±3%) .In a 2004 study conducted in Tehran University, 16% of neonates with NICU hospitalization had hearing loss. Although the authors demonstrated no significant relationship between NICU admission and hearing loss . In 2010In our study, the rate of hearing disorders among NICU neonates was 1.9%; 6.5-times that of RIU babies. In total, 84% of failure cases in screening tests were from RIU babies. This indicates that universal screening programs should be preferred over selective high-risk population screening.