Surfactant proteins (SPs) are important lipoprotein complex components, expressed in alveolar epithelial cells type II (AEC-II), and playing an essential role in maintenance of alveolar integrity and host defence. Because expressions of SPs are regulated by cyclic adenosine monophosphate (cAMP), we hypothesized that phosphodiesterase (PDE) inhibitors, influence SP expression and release. Analysis of PDE activity of our AEC-II preparations revealed that PDE4 is the major cAMP hydrolysing PDE in human adult AEC-II. Thus, freshly isolated human AEC-II were stimulated with two different concentrations of the PDE4 inhibitor roflumilast-N-oxide (3 nM and 1 µM) to investigate the effect on SP expression. SP mRNA levels disclosed a large inter-individual variation. Therefore, the experiments were grouped by the basal SP expression in low and high expressing donors. AEC-II stimulated with Roflumilast-N-oxide showed a minor increase in SP-A1, SP-C and SP-D mRNA mainly in low expressing preparations. To overcome the effects of different basal levels of intracellular cAMP, cyclooxygenase was blocked by indomethacin and cAMP production was reconstituted by prostaglandin E2 (PGE2). Under these conditions SP-A1, SP-A2, SP-B and SP-D are increased by roflumilast-N-oxide in low expressing preparations. Roflumilast-N-oxide fosters the expression of SPs in human AEC-II via increase of intracellular cAMP levels potentially contributing to improved alveolar host defence and enhanced resolution of inflammation. Pulmonary surfactant is composed of a variety of different lipids and four surfactant associated proteins  and is important for reduction of the surface tension at the air-liquid interface of the lung and participates in immunomodulatory processes. Alveolar epithelial cells type II are the main source expressing, synthesizing, and secreting all surfactant components The most abundant surfactant protein in pulmonary surfactant is SP-A. Functional SP-A is important for secretion, synthesis and recycling of surfactant phospholipids and involved in innate immune reactions. SP-D regulates lipid homeostasis and some effects of SP-A. Both, SP-A and SP-D, bind to a variety of pathogens, acting as an opsonin to enhance phagocytosis and support pulmonary clearance. Besides these immune defence tasks, they disclose anti-inflammatory effects and play a role in the resolution of pulmonary inflammation SP-B and SP-C are essential for reduction of surface tension and stabilization of mammalian surfactant lipids SFTPA1 coding for SP-A1 and SFTPA2 coding for SP-A2 and one SP-A pseudogen SP-B, SP-C and SP-D are encoded by single genes It has been shown that in foetal lungs SP-A, SP-B, SP-C and SP-D mRNA are regulated by intracellular adenosine 3̀, 5̀-cyclic monophosphate (cAMP) The current study was designed to unravel potential effects of the PDE4 inhibitor roflumilast-N-oxide on SP-A1, SP-A2, SP-B, SP-C and SP-D in isolated adult human primary AEC-II.The study was approved by the Ethics Committee of the University Medical Centre Freiburg. The material used for isolation of the cells was selected by AK and TP after lobectomy or pneumectomy due to lung cancer. Written informed consent from all participants involved was obtained.In total, macroscopically normal lung tissue samples were obtained from pulmonary resections of 38 patients with primary lung cancer undergoing partial resection, lobectomy or pneumonectomy. The fact that in histological examination all tumours were surrounded by normal tissue suggests that the used samples were free of malignant cells. 25 patients were current smokers; seven patients were ex-smokers and one patient never smoked (5 unknown). Mean smoking duration of the smokers was 39±22 pack-years (14 unknown). Mean age of the patients was 62±10 years. 29 patients were male and nine were female.Based on median relative surfactant protein mRNA expression the donors were defined as “low” or “high” surfactant expressing donors for statistical analysis .7 cells/dish) in RPMI 1640 (Invitrogen) with 10% foetal bovine serum  and 1% penicillin/streptomycin  at 37°C in humidified incubator  for 15, 20 and if necessary 30 minutes to remove adherent  from non-adherent cells.AEC-II were isolated as described previously Non-adherent cells were collected and counted. Cell viability was assessed using the trypan-blue exclusion method. After washing two times, the cells were further separated by anti-CD45-coated magnetic beads  using appropriate depletion columns as suggested by the supplier to remove remaining monocytes/macrophages and lymphocytes. Identity of the negatively fractioned AEC-II was confirmed by a modified Papanicolaou staining. The percentage of Papanicolaou-positive AEC-II was always >90%. The isolated AECII were resuspended and washed one time in culture medium. Preliminary experiments showed a decrease in surfactant protein expression over time, therefore we decided not to exceed 24 h of culture.6 cells·mL–1) were treated with dibutyryl-cAMP (dbcAMP) and roflumilast-N-oxide  with or without 1 µM indomethacin and 10 nM prostaglandin E2 (PGE2) in 24 well plates at 37°C for 24 h. Attempts to measure surfactant proteins by ELISA or to perform Western blot directly from the cell culture supernatant failed. We therefore added 10 µg/ml Brefeldin  to the cell cultures to trap the surfactant proteins within the cells. After supernatant removal, cells were lysed using 350 µl of “RLT plus lysis buffer”  for RNA isolation resp. 200 µl lysis buffer , 2 mM EDTA, 2 mM EGTA, 0.1% Triton-x-100, 1% protease inhibitor mix  for Western blot.AEC-II were cultured in RPMI 1640 with 10% FBS and 1% antibiotics (50 U/mL penicillin and 50 µg/mL streptomycin). To assess the effect of cAMP on surfactant protein expression AEC-II (2×106 cells) were washed twice in phosphate buffered saline (4°C) and resuspended in 1 mL homogenization buffer . Cells were disrupted by sonication and lysates were immediately used for phosphodiesterase (PDE) activity measurements.Human AEC-II cells , 100 µM EGTA, PDE isoenzyme-specific activators and inhibitors as described below and AEC-II lysate. Incubations were performed for 20 min at 37°C and reactions were terminated by adding 50 µl 0.2 M HCl per well. Assays were left on ice for 10 min and then 25 µg 5̀- nucleotidase  was added. Following incubation for 10 min at 37°C assay mixtures were loaded on QAE-Sephadex A25 columns (1 mL bed volume). Columns were eluted with 2 mL 30 mM ammonium formiate (pH 6.0) and radioactivity in the eluate was counted. Results were corrected for blank values (measured in the presence of denatured protein) that were below 2% of total radioactivity. cAMP degradation did not exceed 25% of the amount of substrate added. The final DMSO concentration was 0.3% in all assays. Selective inhibitors and activators of PDE isoenzymes were used to determine activities of PDE families as described previously ++ and 100 nM calmodulin. The increase of cAMP (0.5 µM) degrading activity in presence of 1 µM piclamilast and 10 µM motapizone induced by 5 µM cGMP represented PDE2.The assay mixture  contained 30 mM Tris HCl pH 7.4, 5 mM MgClQuantitative PCR were performed following standard protocols. Total RNA was extracted from AEC-II using RNeasy plus Mini Kit  according to the manufacturer’s protocol. To obtain cDNA, RNA (max. 5 µg) was reverse transcribed using StrataScript reverse transcriptase  with oligo-dT primers  for 1 h at 50°C. Oligonucleotide primers specific for the individual surfactant proteins were designed according to published sequences shown in  and weret) was calculated and used to calculate the relative expression (rE) level of mRNA for each sample by using the following formula:Glyceraldehyde-3-phosphate dehydrogenase (GapDH) was used for normalization. The primer efficiency for GapDH was 96%, SP-A1 112%, SP-A2 85%, SP-B 91%, SP-C 95% and SP-D 100%. A threshold cycle value . Following the stimulation period the cells were washed once in PBS and lysed in ice cold lysis buffer. Whole cell lysates were boiled at 93°C for 5 minutes in equal volumes of loading buffer . Native SP-A purified from a patient with alveolar proteinosis  was used as positive control. All samples were subjected to 12% sodium dodecylsulfate polyacrylamide gel (SDS-PAGE), separated electrophoretically and transferred to a polyvinylidene difluoride membrane . After blocking for 2 h in Tris-buffered saline (TBS) containing 5% non-fat dry milk, the membranes were incubated with primary antibody detecting both isoforms of SP-A  diluted 1∶700, SP-B  1∶200, ProSP-C  1∶400 or SP-D  1∶10000 with TBS at 4°C overnight. After incubation the membrane was washed five times in TBS containing 0.1% Tween 20 (TTBS). For visualization a secondary antibody  was applied at a 1∶10 000-1∶20000 dilution for 2 h. After further five washes, blots were visualized using a chemiluminescent detection system  or using the Odyssey system (Li-COR Biosciences), respectively.To analyze total SP-A expression on protein levels, 3×10Data are presented as mean ± SD. Paired analysis was performed by Wilcoxon signed rank test, unpaired analysis by Mann-Whitney and correlations were performed using Spearman Rank Correlation Test. Probability less than 0.05 values were considered significant. Calculations were performed using STATVIEW 5.01 Version software .First we addressed whether PDE4 is present in primary human AEC-II: To this end lysates of AEC-II from three different donors were analyzed for cAMP- or cGMP-hydrolyzing phosphodiesterase activity in the presence or absence of selective inhibitors to dissect PDE 1–5. Under these conditions, PDE4 impresses as the major cAMP hydrolysing activity  while SP-A1, SP-A2 and SP-C mRNA expression remain constant .AEC-II preparations disclosed a large variation in the mRNA expression of all types of surfactant proteins. Within the non-cultured preparations mRNA expression of SP-A1, SP-A2, SP-C, and SP-D, respectively, correlated significantly and positively with each other . No correlation of SP-B mRNA expression with any of the other surfactant proteins was detected. After culture, mRNA expression of SP-C and SP-D still showed a significant and positive correlation , whereas the correlation of SP-A1 and SP-A2 decreased  and no further correlation could be observed. These data suggest an unequal loss of surfactant protein mRNA expression within the culture period.Highest expression of SP-B in the non-stimulated cultures was found in ex-smokers compared with active smokers  and high baseline intracellular mRNA expression . Regarding cells with a low baseline SP-A1 mRNA expression level, stimulation with roflumilast-N-oxide 1 µM induced an insignificant trend towards augmentation in SP-A1 mRNA expression, but not in SP-A2 mRNA .AEC-II produce PGE2 that may act in an autocrine manner to modulate their cAMP content in a donor-dependent manner, which may in turn influence effects resulting from PDE4 inhibition. To minimize these suspected interactions related to endogenous PGE2 isolated AEC-II were preincubated with the unselective cyclooxygenase inhibitor indomethacin (1 µM). PGE2 blockade indeed reduced SP-A2, SP-B, SP-C and SP-D expression significantly by 50–70% of the controls without Indomethacin whereas the reduction in SP-A1 expression did not reach statistical significance . AdditioRegarding all AEC-II preparations, no increase in SP-A mRNA level  could beApplying the same protocol as described above no significant increase in SP-B mRNA expression could be observed neither in the entire group  nor afteDividing by basal median expression revealed that the overall increase in expression was mostly attributed to the low expressing preparations , p<0.05.Incubation of AEC-II with dbcAMP or roflumilast-N-oxide without indomethacin and PGE2 causes no changes in SP-C mRNA level . HoweverIncubation of AEC-II with dbcAMP or roflumilast-N-oxide without indomethacin and PGE2 causes no changes in SP-D mRNA level . HoweverAs shown above we found striking differences in SP-A mRNA expression in the different cell isolations which should be reflected by differences on protein level. Indeed, also on protein levels some cell preparations showed non-stimulated high while others disclosed low SP-A protein expression. As demonstrated by mRNA expression, SP-A production of high expressing AEC-II increased only slightly after stimulation with dbcAMP or roflumilast-N-oxide  and NF-κB to the TTF-1 binding element (TBE) et al.We and Wang 1A0 found in patients with RDS, is associated with low SP-A mRNA levels For example, human SP-A exhibits a complex regulation at genetic and transcriptional levels. SP-A 1/SP-A2 mRNA ratio varies among individuals from 0.94 to 6.8 A decrease of surfactant proteins in culture is a very early sign of differentiation into AECI-like cells of the cultured AEC-II In our experiments, surfactant protein mRNA expression of AEC-II was differentially increased following incubation with dibutyryl cAMP or roflumilast-N-oxide. The large inter-individual variation might occlude the influence of dbcAMP or roflumilast-N-oxide on surfactant protein mRNA expression. To reduce the variation we grouped AEC-II isolates from the different donors in two groups according to the expression of higher or lower levels of surfactant protein mRNA compared to the median of the entire cohort. However, even after this separation in most cases stimulation of AEC-II with roflumilast-N-oxide or dbcAMP showed no conclusive or only an insignificant up-regulation of surfactant protein mRNA which might be attributed to inter individual variations within endogenous cAMP or PGE2-levels. AEC-II are acknowledged producers of PGE2 which may act autocrinously to augment the cAMP content of these cells Under the conditions described above, stimulation with dbcAMP or roflumilast-N-oxide caused no increase in the respective mRNA expression in AEC-II preparations with a high level of intracellular basal SP-A1 and SP-A2 mRNA expression. However, in cells with low basal intracellular surfactant protein level, in most cases SP-A mRNA expression increased by incubation with dbcAMP and significantly by exposure with roflumilast-N-oxide. Incubation of AEC-II with roflumilast-N-oxide at 3 nM, which is close to its therapeutic plasma levels, resulted in a significant increase in SP-A1 and SP-A2 mRNA in cells with a low intracellular basal level. In conclusion, in human adult AEC-II that display low basal SP-A gene expression, inhibition of PDE4 up-regulates SP-A mRNA expression.In human foetal lung the SP-A2 gene is more sensitive to cAMP than SP-A1 Corroborating our results on mRNA level, Western blot analysis showed also an increased protein level after incubation with dbcAMP and roflumilast-N-oxide (1 µM and 3 nM) in low basal SP-A producing AEC-II. With the exception of SP-D protein, which was not detectable on protein level, similar results were obtained after stimulation with both roflumilast-N-oxide concentrations for SP-B and SP-C protein expression. Thus, the effects of increasing cAMP levels are not only limited to the increase in mRNA but they are also translated in increased protein release.In these experiments we used isolated human primary AEC-II of high and documented purity. The advantage of the use of purified cells is that all effects observed can be attributed to the cell type of interest. Thus, our data demonstrate that roflumilast-N-oxide directly targets AEC-II. In tissue, however, it is possible that the observed effects might be secondary to interactions with other cells; e.g. increase in cAMP in macrophages might induce mediator release which subsequently stimulates alveolar epithelial cells type II. However, this might also be a limitation of our study as in vivo such interactions of AECII with other cells might be normal. A possibilty to include cell-cell interactions might be the use of tissue culture instead of isolated cells and surfactant protein mRNA expression ex vivo might measured either by PCR or using tissue staining or in situ-hybridization Nevertheless, the results of these experiments indicate that inhibition of PDE4, the major common phosphodiesterase in AECII, modulates surfactant protein expression in culture. Regarding the manifold biophysical and immunomodulatory activities of the surfactant proteins, interventions up-regulating surfactant proteins may unfold favourable effects in a range of respiratory ailments given their role in lung physiology and innate defence also taking into account that levels of SP-A were found to be compromised in COPD In conclusion the PDE4 inhibitor roflumilast-N-oxide increased the expression of SP-A and other surfactant proteins in isolated human adult AEC-II expressing lower levels of these proteins at baseline. Considering that cAMP is well described to augment expression of the surfactant proteins in foetal AEC-II, the increase in cAMP following treatment with the PDE4 inhibitor may account for these observations. To our knowledge, this is the first report demonstrating that PDE4 inhibitors regulate surfactant protein expression in human adult AEC-II. This increase in surfactant protein expression was visible in all surfactant protein subtypes, although the increase did not reach a significant level in all cases.Figure S1Surfactant protein mRNA level in AEC-II from current and ex-smokers. Relative expression of SP-B is significantly reduced in ex-smokers  compared with non-smokers . No significant differences between smokers and ex-smokers could be detected in the relative expression of SP-A1, SP-A2, SP-D, and SP-C.(EPS)Click here for additional data file.Figure S2SP-B mRNA level in absence or presence of indomethacin and PGE2. Freshly isolated AEC-II were cultured for 24 h without  or with (C) indomethacin/PGE2 and the indicated substances. After the culture period SP-B mRNA expression was measured by real-time PCR. Due to the large variation in surfactant protein mRNA expression cultures were separated according their basal median expression of SP-B . Bar charts show means ± SD .(EPS)Click here for additional data file.Figure S3SP-C mRNA level with and without indomethacin and PGE2. Cells were incubated 24 h without  or with (C) indomethacin/PGE2 and the indicated substances. After the culture period SP-C mRNA expression was measured by real-time PCR. Due to the large variation in surfactant protein mRNA expression cultures were divided according their basal median expression of SP-C . Values presented are means ± SD .(EPS)Click here for additional data file.Figure S4SP-D mRNA level with and without indomethacin and PGE2. AEC-II were cultured for 24 h without  or with (C) indomethacin/PGE2 and the indicated substances. After 24 h of culture SP-D mRNA expression was measured by real-time PCR. Due to the large variation in surfactant protein mRNA expression cultures were grouped according their basal median expression of SP-D . Bar charts show mean ± SD .(EPS)Click here for additional data file.


In the form of molybdate the transition metal molybdenum is essential for plants as it is required by a number of enzymes that catalyze key reactions in nitrogen assimilation, purine degradation, phytohormone synthesis, and sulfite detoxification. However, molybdate itself is biologically inactive and needs to be complexed by a specific organic pterin in order to serve as a permanently bound prosthetic group, the molybdenum cofactor, for the socalled molybdo-enyzmes. While the synthesis of molybdenum cofactor has been intensively studied, only little is known about the uptake of molybdate by the roots, its transport to the shoot and its allocation and storage within the cell. Yet, recent evidence indicates that intracellular molybdate levels are tightly controlled by molybdate transporters, in particular during plant development. Moreover, a tight connection between molybdenum and iron metabolisms is presumed because (i) uptake mechanisms for molybdate and iron affect each other, (ii) most molybdo-enzymes do also require iron-containing redox groups such as iron-sulfur clusters or heme, (iii) molybdenum metabolism has recruited mechanisms typical for iron-sulfur cluster synthesis, and (iv) both molybdenum cofactor synthesis and extramitochondrial iron-sulfur proteins involve the function of a specific mitochondrial ABC-type transporter. It was thus proposed that molybdate import and distribution are facilitated by sulfate transporters or related systems. The first identified molybdate-specific transporters from Chlamydomonas reinhardtii  represents the cytosolic key enzyme of nitrogen assimilation that reduces nitrate to nitrite . In addiPlant sulfite oxidase (SO) is a peroxisomal enzyme , which e+. At extremely low concentrations of NAD+, molecular oxygen can serve as alternative electron acceptor with simultaneous generation of superoxides (Xanthine dehydrogenase (XDH) requires Moco, FAD, and two iron-sulfur clusters , and itseroxides . As indieroxides . Indepeneroxides . It is sArabidopsis genome encodes the genes AAO1 – AAO4 and the respective proteins form homo- and hetero-dimers with overlapping, but also distinct substrate specificities  has derived from XDH by gene duplication and neo-functionalization  and therficities ,b. The mdormancy . For AAOompounds .N-hydroxylated substances  has been identified in mitochondria of mammals and catalyzes the reduction of bstances . Like ma analogs  and/or iynthesis .Although the physiological functions of some molybdo-enzymes are as yet not fully understood, it is obvious that others hold key positions in essential or at least important metabolic pathways. Any factor that affects one of these enzymes will thus also affect the respective pathway, with effects on Moco biosynthesis and molybdate supply resulting in the pleiotropic impairment of all molybdo-enzyme activities, associated with severe reduction of plant viability or even death of the plant.2+. The inverse phenomenon, an influence of Fe2+ on molybdate uptake, has later been investigated in excised rice roots, which showed increased molybdate uptake capacity in the presence of FeSO4  or heme (NR) besides Moco and FAD. Indeed, when looking in more detail on the more than 50 molybdo-enzymes known in all organisms, then the vast majority also depends on such iron-containing groups . It seemMolybdenum and iron metabolisms also merge at the function of ATM3, a mitochondrial ABC-type transporter that is crucial for the maturation of extra-mitochondrial iron-sulfur proteins . ATM3 exL-cysteine desulfurase domain that decomposes L-cysteine to L-alanine and sulfur (430SH + S2- = > ABA3-Cys430S-SH) and transferred to the Moco-binding C-terminal domain (2(OH) + S2- = > pterin-MoOS(OH) + O2-]. With receiving this type of Moco, the target proteins of ABA3, AO, and XDH, finally gain activity. Interestingly, L-cysteine desulfurases originated from the more ancient iron-sulfur cluster biosynthesis, in which they abstract sulfur from L-cysteine and deliver it to various scaffold proteins, onto which iron-sulfur clusters are assembled prior to insertion into apo-enzymes  responsible for uptake of molybdate at the root:soil interface. A point, which has not been considered before is whether molybdate-binding and/or -storage proteins exist in plants that might shuttle molybdate between organelles and tissues or store it for specific requirements, respectively. Moreover, very little is known about factors controlling the expression of genes and interaction of proteins involved in molybdenum homeostasis, thus requiring further in-depth analysis on the transcriptome, proteome, interactome, metabolome, and ionome level.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.


CDKL5, the different mechanisms regulating its functions, and the connected molecular pathways. Finally, based on the available data we speculate that CDKL5 might play a role in neuronal plasticity and we adduce and discuss some possible arguments supporting this hypothesis.In the last few years, the X-linked serine/threonine kinase cyclin-dependent kinase-like 5 (CDKL5) has been associated with early-onset epileptic encephalopathies characterized by the manifestation of intractable epilepsy within the first weeks of life, severe developmental delay, profound hypotonia, and often the presence of some Rett-syndrome-like features. The association of CDKL5 with neurodevelopmental disorders and its high expression levels in the maturing brain underscore the importance of this kinase for proper brain development. However, our present knowledge of CDKL5 functions is still rather limited. The picture that emerges from the molecular and cellular studies suggests that CDKL5 functions are important for regulating both neuronal morphology through cytoplasmic signaling pathways and activity-dependent gene expression in the nuclear compartment. This paper surveys the current state of CDKL5 research with emphasis on the clinical symptoms associated with mutations in  STK9 (Serine Threonine Kinase 9) [STK9 gene subsequently appeared as CDKL5/STK9 and, eventually, got renamed CDKL5.The kinase CDKL5 (Cyclin-Dependent Kinase-Like 5) was initially identified through a positional cloning study aimed at isolating disease genes mapping to Xp22. Sequence analysis revealed homologies to several serine-threonine kinase genes and identified one protein signature specific for this subgroup of kinases, therefore, leading the authors to name the gene inase 9) . Within A study of the evolutionary conservation of the human CDKL5 protein sequence (GeneBank accession n°. CAI42485) identified several orthologs in vertebrates with large blocks of homology diffused through the whole protein. Fish orthologs diverge the most from the human protein , while nDespite the clear involvement of CDKL5 for proper brain development, as evidenced from the available clinical and molecular data, CDKL5 functions are still far from being understood. This paper examines the current status of CDKL5 research, highlighting the clinical symptoms involving CDKL5, the diverse isoforms so far described and their regulation of expression, the molecular pathways associated with it, and the most urgent studies required to better understand its functions and its possible involvement in neuronal plasticity.CDKL5 were two girls affected by X-linked infantile spasms [MECP2 [CDKL5 mutations occurred mainly in cohorts of patients with RTT syndrome, or a variant of it, with no MECP2 mutations. By comparing the clinical phenotypes of patients with CDKL5 mutations already described in literature, it became clear that almost all of them presented early epilepsy, starting from 10 days to 3 months after birth. Therefore, in the last years, CDKL5 screenings have been extended to cohorts of both genders in which patients were characterized by epileptic encephalopathy. Interestingly, Intusoma et al. [MECP2-negative RTT patients; this score is even increased when RTT-like features are shown as well. In detail, the authors estimated that clinical sensitivity among females with intractable seizures starting before the ages of 12, 6, and 3 months were 4.7, 11.6, and 14.3%, respectively. Stunningly, this value rises to 31.3% when some typical RTT symptoms are included in the phenotype [Even though the first patients mutated in e spasms , subsequs [MECP2 , 5. Becaa et al.  suggesteCDKL5 is in general comparable between several reports [CDKL5 mutations and common to the general clinical manifestation of RTT patients [CDKL5 patients become evident when they get older [CDKL5 patients are generally characterized by residual hand use, poor eye fixation with avoidance of eye contact, visual impairment, and feeding difficulties. Indeed, cortical visual loss has been described by several reports [CDKL5 patients, whereas they are consistently found in Rett syndrome.Albeit few milder cases have been reported, the clinical phenotype of girls mutated in  reports –9. In pa reports , 10–12. patients –9, 12. Wet older . Further reports –16, and MECP2 or CDKL5 emerges from the clinical severity shown by male patients with CDKL5 mutations with respect to those of the same gender carrying MECP2 mutations. Indeed, it is generally accepted that MECP2 mutations causing classic Rett syndrome in females lead to encephalopathy and death in males with a normal chromosome complement within the first years of life. The same mutations, instead, lead to Rett syndrome in XXY backgrounds or in males with somatic mosaicisms. Eventually, mutations with minor clinical relevance in females can lead to RTT in males [CDKL5 mutations [MECP2 mutations are rarely found in males, Liang et al. estimated that, in a cohort of patients affected by epileptic encephalopathy, CDKL5 mutations have a frequency of 5% in males and 14% in females [CDKL5 testing should be considered independently of the gender, they also lead to an important question: why do mutations in an X-linked gene lead to a similar outcome in females and males? Even though no answers are yet available, we believe that, in the future, it will be important to address whether CDKL5-depleted neurons affect the phenotype of wild-type neurons. Anyhow, these results suggest that whereas a major source of the phenotypic variability associated with MECP2 mutations is given by the pattern of X chromosome inactivation, this does not seem to influence the clinical outcome determined by CDKL5. Accordingly, a study describing a male with Klinefelter syndrome  and a large CDKL5 COOH-truncation presenting a phenotype comparable to those of other CDKL5-positive boys suggests that the presence of a wild-type CDKL5 allele and a balanced pattern of X-inactivation does not reduce the severity of the disease [An interesting diversity between patients mutated in in males . On the utations , 18, 19. females . While t disease .CDKL5 gene occupies approximately 240 kb of the Xp22 region and is composed of 24 exons of which the first three  are untranslated, whereas the coding sequences are contained within exons 2-21 . While CDKL5115 is expressed mainly in testis, two recently identified transcripts are likely to be relevant for CDKL5 brain functions [107 in Cdkl5 mouse models of significant relevance for studying CDKL5 functions [The human ons 2-21 . Two splInterestingly, two isoforms selectively expressed in rat neurons and glial cells have recently been described . By westThe CDKL5 protein belongs to the CMGC family of serine/threonine kinases (including cyclin-dependent kinases (CDKs), mitogen-activated protein kinases (MAP kinases), glycogen synthase kinases (GSK), and CDK-like kinases) and is characterized by an N-terminal catalytic domain (amino acids 13–297), homologous to that of the other CDKL-family members. CDKL5 is unique in this family of kinases as it displays an unusual long tail of more than 600 amino acids without obvious similarity to other protein domains but with a high degree of conservation between different CDKL5 orthologs that differ only in the most extreme C-terminus. Besides the ATP-binding region and the serine/threonine protein kinase active site , CDKL5 is characterized by a Thr-Xaa-Tyr motif (TEY) at amino acids 169–171, whose dual phosphorylation is normally involved in activating, among others, kinases of the MAP kinase family. Moreover, putative signals for nuclear import (NLS) and export (NES) have been found in the C-terminus of the protein, as shown in The limited number of CDKL5 targets has not yet allowed drawing a consensus sequence for this kinase; however, the presence of a critical arginine-residue in the kinase subdomain VIII suggests that CDKL5 might be a proline-directed kinase. Moreover, as some of the other CMGC protein kinases, CDKL5 appears capable of auto-phosphorylating its TEY motif .CDKL5 patients, harboring a wide range of pathogenic mutations, have been described so far, including missense and nonsense mutations, small and large deletions, and frameshifts and aberrant splicing  have been addressed through the overexpression of mutated derivatives in nonneuronal cell lines [CDKL5 gene duplications relevant.Almost 90 different splicing . Even thsplicing . By analll lines , 39–41. ll lines –45, it rSo far, no clear genotype-phenotype correlation of CDKL5 mutations has been established. Some reports suggested that mutations in the COOH-terminus originate milder clinical pictures than those caused by mutations in the catalytic domain, but others state that the nature of the mutation does not correlate with the clinical heterogeneity. Accordingly, Weaving et al.  identifiFuture studies, focused on the identification of direct and indirect partners of CDKL5, will help defining its functions and might lead to the identification of modifier genes representing relevant targets for therapeutic approaches.CDKL5/Cdkl5 mRNA is present in a wide range of tissues besides the brain, where the transcripts levels are the highest [CDKL5 mRNAs can easily be detected in tissues such as testis, lung, spleen, prostate, uterus, and placenta, they are barely present, or under detection levels, in heart, kidney, liver, and skeletal muscle. Even if the data regarding CDKL5 protein expression in different tissues are somewhat conflicting, the transcription data have been partially confirmed by western blotting using adult rat extracts [Expression studies in human and mouse tissues have shown that  highest , 3. Indeextracts , 40. To extracts . Cdkl5 expression in adult mouse brain  is grossly identical [Several mechanisms seem to regulate CDKL5 functions and levels. The presence of different CDKL5 splice variants, of which some, as mentioned, appear to be enriched in brain, indicates that alternative splicing is involved in regulating CDKL5 functions. At the functional level, we still need to understand whether the different CDKL5 isoforms have distinct functions. The only difference that has been observed so far is that CDKL5 isoform , whereasdentical , 21.CDKL5 functions seem to be regulated both through its subcellular localization and through its synthesis and degradation. In brain, CDKL5 is initially predominantly cytoplasmic and progressively accumulates in the nucleus, starting from roughly P14 when approximately 40% of total CDKL5 can be detected in this compartment. However, CDKL5 gets significantly translocated to the nucleus only in certain brain areas: in the cerebellum, for example, more than 80% of CDKL5 remains cytoplasmic while in the cortex it is almost equally distributed between cytoplasm and nucleus . Exogeno115 isoform is, in fact, rapidly degraded in a proteasome-dependent manner in transfected cells. Conversely, the human and mouse CDKL5107 isoforms are more stable and their quantity is not significantly increased by the proteasome inhibitor MG132, thus indicating that the very C-terminal region, from amino acid 905, contains signals responsible for this degradation [As mentioned, the degradation of CDKL5 seems also to be involved in regulating its functions. The long CDKL5radation . Why neuCdkl5 functions does not permit to draw a definitive picture, there are several pieces of evidence supporting this hypothesis.Modulation of cellular activity is the force that regulates dendritic growth and morphology during neuronal development, affecting locally the synapses and regulating transcription at the nuclear level. Similarly, activity-induced calcium influx alters synaptic function in mature neurons, modifying synaptic strength and gene expression . TherefoIn recent years, it has become clear that dendrites and spines are dynamic structures that during early postnatal development undergo a significant remodeling necessary for synapse function and plasticity. As development proceeds up to the adulthood, spines continue to be remodeled in response to diverse stimuli such as LTP and LTD; these changes are considered of high relevance for learning and memory.in vitro and in vivo, with neuronal maturation, reaching the highest levels of expression when neurons acquire a mature phenotype and suggesting an involvement of the kinase in neuronal differentiation and arborization [As already mentioned, CDKL5 expression correlates, both rization , 41, 47.In vivo, the kinase shows overlapping functions and affects neuronal migration [The morphology of neuronal extensions and their spines influences the electrophysiology and behavioral output of the cell. Indeed, arborization defects have already been reported in brain disorders, such as RTT and Fragile X, in which experience-dependent neuronal maturation and plasticity are disrupted . Interesigration . Even ifigration . Rac1 beigration . FunctioBDNF, an activity-regulated gene encoding a neurotrophin already involved in several neurological and psychiatric disorders including RTT, induces a transient phosphorylation of CDKL5. The kinase is required for the capability of BDNF to activate Rac1 [A comprehensive knowledge of the stimuli affecting CDKL5 expression and activities will help to understand how its deficiency impacts brain functions. However, so far, only limited information is available. In particular, it has been demonstrated that ate Rac1 . Furtherate Rac1 –54. Of iate Rac1 . These rate Rac1 .in vitro. Indeed, the two human CDKL5 isoforms (CDKL5107 and CDKL5115) immunopurified from cultured cell lines induce the phosphorylation of a recombinant MeCP2, purified from E. coli [in vivo. Anyhow, it must be recalled that several papers suggest that, in neurons, MeCP2 functions as a dynamic epigenetic factor, regulating gene transcription during learning and memory through activity-dependent phosphorylation of specific serine residues [Cdkl5 expression in response to MeCP2 ablation, DNMT inhibition, and histone deacetylase inhibition [As already mentioned, bidirectional signaling pathways link synapses to the nucleus, and, moreover, sensory experiences influence gene expression. Regarding this topic, little evidence suggests that CDKL5 has a role in regulating gene expression. Chen and colleagues  demonstr E. coli , 22, 39. E. coli , 56. It  E. coli . It is sresidues –60. The residues , 62. Intresidues . The conhibition . MECP2 mainly lead to classic RTT, altered CDKL5 activity would determine, among others, certain phosphorylation-dependent MeCP2 dysfunctions, bringing to a subset of RTT symptoms. Other uncharacterized CDKL5 targets could also be deregulated leading to phenotype specifically associated with mutations in CDKL5. However, as already proposed [CDKL5 might also exist, therefore, reinforcing the link between the two proteins. The two models should not be considered mutually or temporally exclusive. Considering all above, the identification of the cytoplasmic and nuclear phosphorylation targets of CDKL5, together with possible transcription events directly or indirectly affected by the kinase, represents an important future milestone towards understanding its neuronal functions.To conclude, we believe that CDKL5-signaling cascades are involved in synaptic plasticity and learning, affecting spines, dendritic branching, and actin cytoskeleton in the cytoplasm and activity-dependent gene expression in the nucleus . Clinicaproposed , a trans


Cdt2 for degradation. In both situations, the substrate-recognizing subunit Cdt2 is detected as a highly phosphorylated form. Here, we show that both caffeine-sensitive kinase and MAP kinases are responsible for Cdt2 phosphorylation following UV irradiation. We found that Cdt1 degradation was attenuated in the presence of caffeine. This attenuation was also observed in cells depleted of ATR, but not ATM. Following UV irradiation, Cdt2 was phosphorylated at the S/TQ sites. ATR phosphorylated Cdt2 in vitro, mostly in the C-terminal region. Cdt1 degradation was also induced by DNA damaging chemicals such as methyl methanesulfonate (MMS) or zeocin, depending on PCNA and CRL4-Cdt2, though it was less caffeine-sensitive. These findings suggest that ATR, activated after DNA damage, phosphorylates Cdt2 and promotes the rapid degradation of Cdt1 after UV irradiation in the G1 phase of the cell cycle.The DNA replication-licensing factor Cdt1 is present during the G1 phase of the cell cycle. When cells initiate S phase or are UV-irradiated, Cdt1 is recruited to chromatin-bound PCNA and ubiquitinated by CRL4 Ordered progression of the cell cycle is controlled by the periodic activation of cyclin-dependent kinases (CDKs) Skp2-mediated pathway Cdt2) was subsequently shown to be central to Cdt1 proteolysis in yeast to mammalian cells Cdt2-mediated ubiquitination. PCNA connects Cdt1 and CRL4Cdt2 on the chromatin. Interestingly, Cdt1 is degraded after DNA damage, such as UV irradiation, by the same PCNA-mediated CRL4Cdt2 pathway Cdt2 are rapidly recruited to the damaged sites dependent on the chromatin association of PCNA Xenopus egg extracts demonstrated that either the initiation of replication or incubation with damage-containing DNA triggers chromatin loading of PCNA, the association of Cdt1 with PCNA through its PIP box, and the recruitment of Cdt2 Cdt2 pathway include p21, Xic1, and Set8 in vertebrates Cdt2 pathway Recent data demonstrated that ubiquitin-mediated proteolysis is essential for ensuring that DNA replication occurs only once per cell cycle Cdt2-mediated degradation of Cdt1 UV irradiation induces helix-distorting DNA damage such as cyclobutane pyrimidine dimers and 6-4 photoproducts, which trigger several signaling cascades to provoke a cellular response that includes a DNA damage-induced checkpoint response. A replication block due to lesions during the S phase triggers the efficient activation of ATR in vitro, and Cdt2 that was recovered after UV irradiation contained phosphorylated S/TQ sites. These findings suggest that the checkpoint response is involved in the rapid degradation of Cdt1 after DNA damage.Cdt2 contains seven WD40 repeats in the N-terminal half part, which is conserved from yeast to mammals and is thought to form a substrate-recognizing propeller structure. In contrast to yeast, Cdt2 of higher eukaryotic cells has a long C-terminal region. We previously demonstrated that Cdt2 was highly phosphorylated following UV irradiation. Here, we examined whether any kinases regulate Cdt1 degradation following UV irradiation. CRL4-Cdt2 mediated Cdt1 degradation was independent of ATR/ATM We previously reported that the phosphorylation levels of Cdt2 protein are low during the G1 phase when Cdt1 is present at high levels, but that as cells enter the S phase or when cells are exposed to UV irradiation, the Cdt2 phosphorylation levels increase and Cdt1 is degraded 2 and retarded forms of Cdt2 were detected. In contrast, in the presence of caffeine, Cdt1 degradation was delayed and the retarded form of Cdt2 was not clearly detected  and examined the rate of Cdt1 degradation. In control siRNA-treated cells, Cdt1 was degraded to 50% within 30 min. In ATR-depleted cells, Chk1 phosphorylation levels were reduced and Cdt1 degradation was delayed similar to that in caffeine-treated cells . Such a Cdt2 has several SQ/TQ motifs that are found in ATM/ATR substrates. The above results suggested that checkpoint kinase ATR was activated to degrade Cdt1 following UV irradiation, so we next examined if ATR was directly involved in Cdt2 phosphorylation. We isolated a HEK293 cell line that stably expresses Cdt2-FLAG with about five times more Cdt2 protein than that in control cells . The tag32P was evident at the Cdt2/Cul4A position, but not at DDB1, when the complex was incubated with wild-type ATR , an alkylating agent, and zeocin, a gamma radiation-mimicking reagent that creates strand breaks, including double-strand breaks. Both of these reagents induced Cdt1 degradation, depending on the PCNA-CRL4radation . We thenradation . In zeocradation , and theradation . Phosphoradation . Althougin vitro kinase assay demonstrated that Cdt2 protein was phosphorylated by ATR, and Cdt2 isolated following UV irradiation contained phosphorylated S/TQ sites. Human Cdt2 has nine SQ sites and quantitative phosphoproteomic analyses reveal its phosphorylation at several of these sites Licensing factor Cdt1 is rapidly degraded within minutes following UV irradiation. Here, we demonstrated that checkpoint kinase ATR is required for this rapid degradation of Cdt1. The phosphorylation levels of Cdt2, a substrate receptor of CRL4 ubiquitin ligase, is normally low during the G1 phase. Its levels increase not only when cells enter the S phase, but also when cells are irradiated with UV, and Cdt1 is degraded in both cases. The present findings suggest that both MAP kinases and ATR are involved in Cdt2 phosphorylation following UV irradiation. Importantly, ATR, but not ATM or MAP kinases, is required for the rapid degradation of Cdt1. An Cdt2ATR activation following UV irradiation was reported in the S phase in vitro kinase assay also indicated that ATR phosphorylated mostly the C-terminal part of Cdt2. Phosphorylation may modulate the affinity between PCNA, Cdt1, and CRL4Cdt2 in conjunction with DNA on the chromatin, and increase the activity of CRL4Cdt2. We examined whether the chromatin association of Cdt2 was affected in the ATR-depleted cells, but observed no change in the affinity to chromatin (data not shown). Recently, many factors necessary for Cdt1 degradation after UV irradiation were reported through genome-wide screening, such as NER factors, COP9/signalsome, and p97 AAA+-ATPase How does ATR-mediated phosphorylation of Cdt2 promote Cdt1 degradation? In contrast to fission yeast Cdt2, higher eukaryotes have Cdt2 with an extended C-terminal region. As indicated by quantitative phosphoproteomic analyses, phosphorylation is observed mainly in the C-terminal part. Our DNA damaging chemicals, MMS and zeocin, also induced Cdt1 degradation. We observed that caffeine reduced the rate of Cdt1 degradation and that Cdt2 was phosphorylated at S/TQ sites following MMS treatment, suggesting that ATR also promotes Cdt1 degradation in this case. However, the effect of caffeine on Cdt1 degradation and the levels of p-S/TQ was lower than those observed after UV irradiation. Following zeocin-treatment, we detected no p-S/TQ phosphorylation on Cdt2 (data not shown) and caffeine had almost no effect on the rate of Cdt1 degradation . These rMany anticancer drugs cause DNA damage and induce Cdt1 degradation. A recent report demonstrated that anticancer reagents induced Cdt1 degradation, but the levels of response appeared to be cell specific 2. To synchronize cells in the G1 phase, cells blocked in the early S phase using the thymidine and aphidicolin block method were released for 16 h or nocodazole-arrested cells were released for 3 h. Caffeine was used at 10 mM. MAP kinase inhibitors were used at the following concentrations: ERK inhibitor U0126  at 10 µM, JNK inhibitor II  at 30 µM and p38 inhibitor SB202190  at 20 µM. Proteasome inhibitor MG132 was used at 25 µM. MMS was used at 0.5 to 5 mM. Zeocin was used at 2–6 mg/ml. UV-C (254 nm) irradiation of whole cells in dishes was performed at 20 to 100 J/m2 using a UV cross-linker . To analyze the DNA content, flow cytometry was performed as described previously HeLa, HEK293 and HEK293 cells stably expressing Cdt2-FLAG, and HEK293T cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and 5% COFor Western blotting, whole cell lysates were prepared by lysing cell pellets directly in SDS-PAGE buffer. The following primary antibodies were used: Cdt1 rabbit, ; Cdt2 r; ATR , the supernatants were mixed with anti-FLAG antibody-conjugated magnetic beads  to obtain immunoprecipitates from the supernatants. The precipitates were resuspended in 0.1% Triton X-100 mCSK buffer and sonicated. After centrifugation , the supernatants were used as the chromatin-bound fraction for immunoprecipitation with anti-FLAG antibody-conjugated magnetic beads. Protein expression of FLAG-DDB1 and Cdt2 in Sf21 cells with baculo virus system , and purification with anti-FLAG column  was as described Control or Cdt2-FLAG expressing HEK293 cells that were UV-irradiated or not UV-irradiated were lysed using 0.1% Triton X-100 mCSK buffer: 10 mM Pipes, pH 7.9, 100 mM NaCl, 300 mM sucrose, 0.1(v/v)% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 10 mM β-glycerophosphate, 1 mM NaCGGUGACACUCAGUAUGUC, Cdt2; CCAGGAGGUGAUAAACUUU, ATR; CCUCCGUGAUGUUGCUUGA, ATM; GCGCCUGAUUCGAGAUCCU. The siRNA for siLuc, known as GL2, was used as a control siRNA.The double-stranded RNAs were transfected at 100 µM using Oligofectamine (Invitrogen) or HiPerFect (Qiagen). Twenty-four hours after the first transfection, the second transfection was performed and cells were cultured for 2 d. The following siRNAs were made by Dharmacon: PCNA; The assay was performed as described previously


Objective: To investigate the effect of sensitivity to temporal fine structure (TFS) on subjective measures of hearing aid outcome.Design: Prior to receiving hearing aids, participants completed a test to assess sensitivity to TFS and two self-assessment questionnaires; the Glasgow Hearing Aid Benefit Profile (GHABP), and the Speech, Spatial and Qualities of hearing (SSQ-A). Follow-up appointments, comprised three self-assessment questionnaires; the GHABP, the SSQ-B, and the International Outcome Inventory for Hearing Aid Outcomes (IOI-HA).Study sample: 75 adults were recruited from direct referral clinics.Results: Two thirds of participants were found to have good sensitivity to TFS; listeners with good sensitivity to TFS rated their hearing abilities higher at pre-fitting (SSQ-A) than those with poor sensitivity to TFS. At follow-up, participants with good sensitivity to TFS showed a smaller improvement on SSQ-B over listeners with poor sensitivity to TFS. Among the questionnaires, only the SSQ showed greater sensitivity to measure subjective differences between listeners with good and poor sensitivity to TFS.Conclusions: The clinical identification of a patient's ability to process TFS information at an early stage in the treatment pathway could prove useful in managing expectations about hearing aid outcomes. Presbycusis is characterized by gently-sloping high-frequency hearing loss. It is often first revealed to the listener through a reduced understanding of conversational speech, particularly when there is a source of background noise. A common treatment for presbycusis is provision of hearing aids. Hearing aids make understanding speech much easier for the vast majority of people in a range of situations. However, listening in complex or noisy environments can remain challenging for some people even after provision of amplification  for frequencies lower than 1000 Hz because TFS information is thought to be important for the perception of F0 information  followed General Practice (GP) direct-referral route to audiology, (b) 50+ years of age, (c) bilaterally symmetrical sensori-neural hearing loss, (d) had not previously worn a hearing aid, (e) normal or corrected-to-normal vision. Our sample comprised 75 adults (44 men and 31 women) with a mean age of 72.24 ± 0.82 (range age 51–85 years) with mild-to-moderate hearing loss. Ethical approval for this study was obtained from the Derbyshire Research Ethics Committee.Participants were tested by a member of the research team on three occasions. The first testing session took place prior to the patient being fitted with a hearing aid. During this session, participants completed a short test to determine their sensitivity to TFS and a number of self-report assessment questionnaires. The second and third research appointments took place 3- and 6-month post-hearing-aid fitting, in which participants completed a number of self-report outcome questionnaires.Listening to the television with other family or friends when the volume is adjusted to suit other people”) and four self-nominated listening scenarios which allows the listener to specify additional listening circumstances of importance and relevance to their everyday communication circumstances . Higher ratings on each of these dimensions indicate greater levels of difficulty or worry. The SSQ-A asks participants to rate their listening abilities using an ordinal scale (0–10) on three sub-scales: Speech, Spatial, and Qualities on 14, 17, and 18 pre-specified listening scenarios respectively  Higher ratings on the SSQ-A indicate greater levels of perceived ability.In order to ascertain the degree of difficulty experienced prior to provision of a hearing aid in a range of listening scenarios, all participants were asked to complete the first part of the Glasgow Hearing Aid Benefit Profile  guidelines  by a quaAAAA or ABAB sequences. In AAAA intervals, all the tones were presented diotically. In ABAB intervals, the first and third tones were diotic whilst the 2nd and 4th tones were presented with an IPD (ΔØ). Participants were asked to identify which interval contained the tones that appeared to change in location. A two-up, one-down adaptive procedure was used to vary ΔØ. At the beginning of a run ΔØ was set to a maximum value of 180°. Thresholds were calculated by measuring the geometric mean of ΔØ at the last six turn points which corresponded to the 71% correct point. However, the adaptive procedure terminated early if this maximum value was reached twice before the second turn point, or at all after the second turn point. In this situation, the program reverted to a non-adaptive (method of constant measures) procedure in which a further 40 trials were presented with ΔØ fixed at its maximum value and a percentage correct score was calculated. Discriminability index (d′) values for the TFS test were calculated using a table of d′ values for two-alternative forced choice procedures .All hearing assessments were conducted in a double-walled, sound proof boothpost-hoc paired-sample t-tests, One-Way ANOVA and Pearson correlations. Calculation of discriminability index (d′) values for the TFS is described in section Hearing assessments, TFS/0.5 kHz.The statistical methodology employed in this study includes basic descriptive analysis, t = −9.12, p < 0.001]. Audiogram for left and right is shown in Figure Although hearing thresholds were classified to be bilaterally symmetric, the six-frequency pure-tone average hearing thresholds measured at the better ear and the poorer ear (34.5 ± 1.04 dB HL at the better ear) were found to be significantly different  and less worrying  than the self-nominated listening situations  on the Initial Disability sub-scale and “Moderate” levels of worry on the Handicap sub-scale 2.9 ± 0.78). Initial Disability and Handicap scores were strongly correlated with one another, but were not correlated with age or audiometric threshold  = 31.43, p < 0.01].Altogether, 49 participants completed the TFS-LF task using the adaptive procedure while 26 participants reverted to the method of constant measures . Sensitivity to TFS information was confirmed by comparing discriminability index (d′) values for the two groups  = 7.23, p < 0.01] than participants with poorer sensitivity to TFS  was weakly associated with age, and moderately associated with self-report scores of the Spatial and Quality sub-scales of the SSQ-A  = 2.89, p = 0.08], Residual Disability , and Satisfaction .The mean GHABP part two self-report scores for Usage, Benefit, Residual Disability, and Satisfaction are shown in Figure There was no association between GHABP self-reported outcomes and severity of hearing loss, or sensitivity to TFS information. Significant associations are described in Table Participants reported moderate improvements in listening ability on all three sub-scales of the SSQ-B . See Table F = 4.22, p < 0.05] sub-scale. It can be seen from Figure Participants with poor sensitivity to TFS (constant-measures TFS group) reported experiencing greater levels of improvement on all three of the SSQ-B sub-scales. Ratings of improvement were significant different between the two groups on the Qualities , and IOI-HA Residual Activity Limitations decreased during the same period . Participants with the poorest sensitivity to TFS continued to experience better outcomes (SSQ-B) at the 6-month follow-up than those with good sensitivity to TFS .Differences in outcome reported at the 3- and 6-month follow-up appointments were compared using In this observational case series, we monitored the auditory rehabilitation of 75 older adults for a period of 6-months following receipt of their first hearing aid. All patients received standard audiological management pathways  for sensori-neural hearing loss. No experimental interventions or treatment groups were used. However, patients did complete a non-standard pre-fitting assessment to determine sensitivity to TFS information, and a range of non-standard pre- and post-fitting self-report questionnaires. The main purpose of this study was to assess how sensitivity to TFS information contributed to the hearing difficulties that the group faced pre- and post-provision of hearing aids.It is generally accepted that speech perception deteriorates with increasing age  and Qualities of hearing  than participants with poor sensitivity to TFS on the SSQ-A. However, participants with poor sensitivity to TFS reported experiencing greater improvements on the Spatial and Qualities of hearing dimensions of the SSQ-B than the participants with good sensitivity to TFS at the 3-month follow-up, and Speech and Qualities of hearing at the 6-month follow-up. These results suggest that listeners with poor sensitivity to TFS may experience poorer spatial and qualities of hearing than listeners with good sensitivity to TFS prior to their hearing aid fitting, and therefore, experience by contrast greater hearing aid benefit as their initial score was lower and consequently had more “opportunities for improvement” than listeners with good sensitivity to TFS information. Given these differences in self-reported listening abilities and the relative independence of TFS sensitivity and audibility, it appears that an assessment of a patient's sensitivity to low-frequency binaural TFS information could prove useful in managing the expectations of patients who are due to receive a hearing aid. Differences in expectations could, at least partly, explain the observed patter of results in which patients with high sensitivity to TFS may have higher expectations, and consequently more difficult to fulfill, while patients with poor sensitivity to TFS may have lower expectations, easier to fulfill. Perhaps with better management options that take sensitivity to TFS information into account this advantage could be increased further. For instance, there is some evidence to suggest that choice of compression algorithm could be informed by knowledge of a patient's ability to process TFS information  were highly associated with one another, they were not associated with age, severity of hearing loss or GHABP post-fitting outcomes. At the 3-month outcome assessment the four GHABP outcome dimensions  were strongly associated with one another, but again largely independent of age and severity of hearing loss. Moreover, there was a striking dichotomy between self-reports obtained on the pre-defined and self-nominated listening scenarios. The individual needs that may arise when measuring hearing aid benefit across different domains can be better captured when the hearing aid user is giving the opportunity to self-nominate specific scenarios. Those self-nominated scenarios may be very specific and only relevant to a single hearing aid user. While the GHABP is sensitive for capturing those meaningful and idiosyncratic listening difficulties, our results showed that those dimensions are not associated to TFS. These findings limit the efficacy of the GHABP as an outcome tool, at least when comparing group data, but highlight its sensitivity in characterizing patient's needs and therefore treatment improvement . The SSQ-A and SSQ-B, on the other hand, showed high levels of consistency between pre- and post-fitting assessments, and were moderately correlated with GHABP pre-fitting dimensions; the SSQ questionnaires were also the only ones, in this study, to reveal subjective differences between listeners with good and poor sensitivity to TFS. It has been reported that the severity of hearing loss is associated with the amount of hearing aid benefit and satisfaction (Walden and Walden, In the current study, our hypothesis was that those patients with good sensitivity to TFS would have significantly better outcomes than those with poor sensitivity. Our results show that assessing sensitivity to TFS information could prove important to the management of the expectations of first-time hearing aid users. We found that, new hearing aid users with good sensitivity to TFS reported experiencing less debilitating hearing difficulties prior to provision of hearing aids, but also reported experiencing the least amount of improvement following provision of hearing aids compared to listeners with poor sensitivity to TFS. The TFS test employed in this study (TFS-LF: Hopkins and Moore, 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.


Neonatal hypernatremia dehydration (NHD) is a dangerous condition in neonates, which is accompanied by acute complications  and chronic complications . Children begin learning language from birth, and hearing impairment interferes with this process. We assessed the hearing status of infants with hypernatremia dehydration.In a case-control study in 110 infants presenting at the Ghaem Hospital  between 2007 and 2011, we examined the incidence of hearing impairment in infants suffering from hypernatremia dehydration (serum sodium >150 mEq/L) in comparison with infants with normal sodium level (serum sodium ≤150 mEq/L).Three of 110 cases examined in the study group showed a transient hearing impairment. A mean serum sodium level of 173mg/dl was reported among hearing-impaired infants.Transient hearing impairment was higher in infants with hypernatremia; although this difference was not significant (P>0.05). Hearing impairment was observed in cases of severe hypernatremia. Neonatal hypernatremia dehydration (NHD) is a potentially dangerous condition often caused by inadequate nutrition during the first week of life . NumerouThis case-control study was conducted on 1–29-day old infants presenting to the Ghaem Hospital and those hospitalized at the neonatal intensive care unit (NICU) and Emergency Ward. The case group consisted of 50 neonates hospitalized at the NICU and infants with sodium levels higher than 150 mg/dl, and the control group consisted of 65 infants brought to the hospital for routine check-up or physiological jaundice, with sodium levels lower than 150 mEq/L, and bilirubin levels lower than 15 mg/dl. Infants with hearing impairment of any origin and those with its risk factors were excluded from the study. On enrollment, a checklist consisting of the following items was completed: demographics, birth weight, current weight, pregnancy age, the Apgar score, duration of hospitalization, presence/absence of anomalies, symptom at hospital admission, breastfeeding status, feeding types (using or not using supplements), frequencies of urination and defecation, breast condition and problems, mother’s age, mother’s medical history and delivery method. Laboratory data  and para clinical tests  were reported.Hearing status based on OAE and ABR tests Infants in both groups underwent an otoacoustic emissions (OAE) test by an audiologist in addition to an ear examination. Infants with abnormal OAE underwent a further OAE test at the age of one month; and if the abnormality persisted, they underwent an auditory brainstem response (ABR) test. In case of ABR abnormality, this test was repeated every three months. We then examined and compared hearing impairment incidence in NHD neonates and neonates with normal sodium levels. The data were analyzed with SPSS 16, and P<0.05 was considered a significant value.In this study, questionnaires were completed for 120 neonates, two of whom were excluded for a family history of hearing impairment, one for otitis media, and seven because their parents did not return for follow-up; hence, 110 neonates were studied.In the case group, the mean duration of NHD was nine days, and the mean weight loss was 11.7%. The peak incidence in the case group was at the sixth day after birth. The chief cause of admission included poor feeding and fidgetiness, followed by lethargy, hyperthermia, and jaundice. The on-admission complaints and findings in the case group after weight loss (60%) were poor feeding 44%, hyperthermia 38%, fidgetiness 34%, lethargy 32%, mucosal dryness 16%, seizure 14%, jaundice 14%, apnea 4%, decline in consciousness 2%, and cyanosis 2%. In our study, most mothers were between 23 and 26 years of age, and the highest incidence was found in the first parity (66%). In our study, there was no significant relationship between the incidence of hypernatremia and the time of first breastfeeding, the mother’s age, pregnancy complications or duration of breastfeeding (P>0.05), but there was a significant relationship between the incidence of hypernatremia and the use of supplements in addition to breastfeeding, frequency of feeding and frequency of urination (P<0.05). The mean feeding frequency was 7 and 12 occasions in 24 hours in the case group and control group, respectively. Cerebral problems occurred in 8% of the neonates, three of whom had cerebral edema and one, a cerebral hemor- rhage.In the case group, three neonates had an abnormal OAE at the onset of hypernatremia, followed by a normal OAE at the age of one month. Thus, the incidence of hearing impairment in this study “at the onset of hypernatremia” was 0% in the control group and 6% in the case group, and the statistical difference was not significant (P>0.05). The mean plasma sodium level in neonates with hearing impairment was higher than among controls .Hypernatremic neonates with hearing impairment had a lower serum potassium and a higher creatinine compared with neonates with normal hearing (P<0.05). The former also had a lower mean blood sugar and higher age, although these differences were not statistically significant .Hearing impairment is an asymptomatic disability in children, as speech development in children is delayed secondary to hearing impairment. Any delay in the diagnosis of hearing impairment can lead to adverse and irreversible consequences for the child ,5. UnforIn our study, the age of the children in the control groups was three days younger than that in the case group (6±3.6 as opposed to 9±6.6 days), which shows that NHD infants are admitted towards the end of their first week. Likewise, in a study by Gomes , the meaIn our study, there was no significant relationship between the incidence of hypernatremia and the time of first breastfeeding, mother’s age, pregnancy complications, or breastfeeding duration (P>0.05), but there was a significant relationship with number of feeding times per day (P=0.00). In others studies, there was also no relationship between the incidence of hypernatremia and the time of first breastfeeding ,11. In sIn this study, the most frequent neonatal problem in the case group was weight loss; which was severe (over 10%) in 60% of the neonates. As for other symptoms, the most frequent on-admission complaints and findings in the case group after weight loss (60%) in view of accompanying findings in the case group were poor feeding (44%), hyperthermia (38%), fidgetiness (34%), lethargy (32%), mucosal dryness (16%), seizure (14%), jaundice (14%), apnea (4%), reduced consciousness (2%), and cyanosis(2%).In this study, the range of sodium levels in the case group was 150–200 mEq/L and in the control group, 131–149 mEq/L. The mean sodium level in the case group was 165.7±12.9 mEq/L and in the control group, 141.18±4.71 mEq/L. The range of sodium levels in our study was similar to that in others study , which is similar to a study by Morris-Jones et al, in which 12 infants 37%) had abnormal neurological examinations and 9% suffered from major neurological conditions . In the 7% had abIn this study, none of the infants had abnormalities in the OAE in the control group. In the case group, three infants had abnormal OAE at the onset of hypernat- remia, but their subsequent OAE at the age of one month was normal. Thus, the incidence of hearing impairment in this study “at the onset of hypernatremia” was zero percent in the control group and 6% in the case group, a statistically insignificant difference (P=0.09). Therefore, there is no statistically significant relationship between NHD and hearing impairment. Noneth- eless, this may be an artifact of the small size of the sample in this study. It should be noted that the incidence of hearing impairment in NHD infants in our study was 60 times greater than that reported worldwide, which seems like a significant difference.Of the three hearing-impaired infants, two had severe hypernatremia and one mild hypernatremia, and the mean plasma sodium level in the hearing-impaired infants was greater than in infants with normal hearing . Therefore, it can be concluded that hearing impairment occurs with high levels of sodium. Likewise, in the study by Pasajlu and the study by Morris-Jones et al, hypernatremic complications occurred with higher levels of sodium. These complica- tions were also more frequent with higher levels of blood urea . Also inIt should be noted that changes in the endolymph and perilymph in the middle ear are not independent of the body’s hemodynamics, as can be seen in Meniere's disease. In this disease, endolymphatic hydrops causes dizziness, tinnitus, and fluctuated sensorineural hearing loss. Certain studies have recognized the contribution of the sudden combination of the perilymph and endolymph due to the rupture of the membranous labyrinth to this disease, which leads to physical and chemical changes in the vestibulocochlear system . DiuretiOverall, it can be concluded that hypernatremic dehydration, especially in severe cases, can lead to transient abnormalities in audiometric tests, although in our study there was no significant statistical difference between the case and control groups in terms of the incidence of hearing impairment, which may be due to the small size of the sample in this study.


Increasingly, our acoustic environment is filled with amplified sound sources . There is serious concern and also some controversy about the risks of acoustic trauma in children. This overview provides some basic information on the physiological mechanisms that lead to noise induced hearing loss, a survey of various studies that, on balance, indicates that there is cause for concern, and finally a discussion on measures that can help to prevent noise induced hearing loss in children. This paper is designed for public health and other healthcare professions  who should understand the risks of noise induced hearing loss and its prevention. In most of our societies (both developed and developing) there are increasing levels of acoustic signal exposure, in large part due to the (electronic) amplification of sound and to the ubiquitous use of audio entertainment devices. Attention has shifted from the noise exposure problems in specific groups, for example, in industrial or military environments, to a more widespread potential source of noise trauma. A variety of sound sources, for example, earphones and in-door and outdoor loudspeakers, are capable of decibel levels that can result in acoustic trauma if not used carefully. This paper is provided for public health and hearing healthcare professionals so that they can more fully understand the problem of noise induced hearing loss and provide authoritative advice. A range of the literature is reviewed including epidemiological studies, data from hearing related questionnaires and surveys, audiology case studies, and reports of (noise induced) tinnitus. This paper also provides advice on how to prevent noise induced hearing loss, both for patients/parents and in educational programs for school children. To start, here is an overview of physiological mechanisms involved in acoustic damage to the cochlea.Many of the earliest auditory science studies were carried out to explore the effects of acoustic trauma, for example, going back to work by Davis and colleagues in 1935 . There iVery intense acoustic signals (>130 dB SPL) can directly cause mechanical damage to the cochlea as well as to middle-ear structures. The initial insult may not be directly to hair cells, but to other supporting structures in the organ of Corti as well as Reissner's and tectorial membranes. Hair cell and neuronal degeneration may follow subsequently, perhaps due to mixing of endolymph and perilymph or the release of cytotoxic agents  from damaged tissue. In the context of “recreational” noise exposure , it is unlikely that stimuli will reach levels to cause direct lesions to the organ of Corti. Less intense acoustic signals, especially if impulsive (high concentration of energy in the time domain) or with dominant spectral energy peaks  can mechanically damage hair cells, with the most vulnerable structures being the stereocilia.Milder acoustic exposure for a long duration can cause intracellular changes in hair cells related to, for example, metabolic depletion or excessive release of neurotransmitter. Up to a certain point, metabolic damage in hair cells is reversible, and as such may be manifested in a recovery from a temporary threshold shift. However at some tipping point, apoptosis (programmed cell death) will occur and lead to complete hair cell loss. The story does not end here. Any damage at the cochlear level leads to central auditory pathways alterations. Most locally, excess release of glutamate from overstimulated inner hair cells can cause excitotoxicity to the cochlear afferent neurons. With such deafferentation, degenerative changes can be observed in second- and third-order neurons throughout the auditory brainstem and midbrain. Some of these pathophysiological events are discussed in more detail below.A number of ultrastructural changes to cochlear hair cells resulting from noise exposure can be attributed to metabolic exhaustion –5. TheseThe stereocilia are key elements in the mechanoelectrical transduction process, and the effects of noise trauma on these structures have been extensively studied. Scanning electron microscopy has proved a particularly useful tool e.g., –13).  Fi.  Fi13])Examples of stereociliar damage caused by acoustic trauma are shown in The images of What cochlear pathology is reversible and might therefore underlie temporary threshold shifts? What damage is irreversible and related to permanent threshold shift? There is some evidence that very mild damage to stereocilia can recover , 26, 31.Pioneering studies on the effects of noise trauma involved the recording of gross electrophysiological potentials, such as the cochlear microphonics and compound action potentials, to monitor cochlear function e.g., , 32). Th. Th32]).Liberman and his colleagues –26 have Experiments that have investigated the long-term effects of acoustic trauma on cochlear neural responses reveal the chronic functional changes that are likely present in patients with noise induced hearing loss. This loss of cochlear frequency selectivity translates into an inability of the auditory system to analyze frequency components in complex sound and is, thus, a contributing factor to poor speech intelligibility in patients with moderate to severe hearing loss from acoustic trauma e.g., –42)..42]).In addition to the direct trauma that intense sounds can cause to hair cells, there are also secondary effects that can cause further damage. Just as with a brain injury , there can be initial restricted lesion, but then cellular byproducts that are released, for example, oxidative free radicals or excessive amounts of neurotransmitter, can cause more extensive further damage. It is also possible that prolonged acoustic overstimulation can lead to local vascular damage and a cochlear hypoxia that in turn can cause damage to hair cells .One issue that has been of some interest to clinicians is the question of whether medical treatment with steroids  has any utility in treating acute sensorineural hearing loss, including noise induced sudden deafness. The concept behind such treatment is that the progression of damage that comes subsequent to the initial hair cell damage (as outlined above) may be prevented. This issue has been controversial for some decades, and very recent discussion continues with an editorial by Piccirillo in JAMA . CommentOther than the loss of hearing sensitivity, there are two other common symptoms related to noise induced hearing loss. One is temporary threshold shift; the other is tinnitus or ringing in the ears. Regarding the former, after exposure to a period of loud sound, there can be a “temporary” mild hearing loss. We have all experienced this after a long air flight or bus journey or following a loud music concert. We tend not to worry about such experiences, in part because there appears to be a full recovery. However, it is widely supposed that repeated episodes can result in permanent changes. As we reviewed above, it is likely that the noise exposure resulting in temporary threshold shift alters the delicate micromechanics of the cochlea or the structure of stereocilia and their delicate linkages see . For botThere are a number of types of tinnitus, and not all result from cochlear damage. Often, tinnitus is transient, and indeed it is normal to occasionally hear a brief ringing in the ear that dies away within a few seconds. However, when chronic tinnitus is experienced after exposure to loud sounds, it is not just a warning sign but a clear manifestation of cochlear injury. Consider the ringing sound to be caused by hair cells and neurons actually in the process of dying. Such cells generate a neural injury discharge because the cell membrane breakdown causes repeated depolarization (excitation) and/or uncontrolled release of neurotransmitter. In the case of a severe acoustic trauma, tinnitus can persist and become permanent. It has been suggested that the initial neural injury discharge sets up and/or strengthens synaptic connections in a network of auditory neurons at a more central  brain level and that these cells continue to fire spontaneously. This could be because a local positive feedback circuit is established or because local inhibitory neuron activity is reduced . ChronicWe are all born with a fixed number of cochlear hair cells. In humans , they do not regenerate, and so we should take preventative care of these cells. In some vertebrates, for example, in birds, hair cells do regenerate after damage. Actually new hair cells develop from local supporting cells that act like a type of stem cell. In our vestibular sense organs, the hair cells are capable of regeneration, but in the cochlea this does not happen. There is presently a considerable research effort to determine if cochlear hair cells can be made to regenerate, either by providing suitable growth hormones or by finding a genetic switch to turn on the cell differentiation process e.g., –51). Pre. Pre51])Here is a survey of some of the current literature on the noise induced hearing loss in children and young adults. This is not an exhaustive or systematic review, but a representative sample of studies which all point to the growing problem. It has to be recognized that in this area, there is no definitive, “level A” evidence, that is, prospective, randomized controlled studies. However, there are numerous other study types, most of which are cautionary.In terms of general population studies, a report from a large-scale US national health survey indicates that 12–15% of school-age children have some hearing deficits attributable to noise exposure . In CanaN = 24) of German teenagers [There are many smaller scale studies addressing noise induced hearing loss in children. In a Scandinavian research , hearingeenagers .A number of studies have specifically focused on possible hearing loss from personal music players. In an interview study of 490 Korean adolescents, a strong conclusion was that long-term exposure to music player can have “a deleterious effect on hearing thresholds.” The authors ascribe the hearing losses to the use of personal entertainment devices and attendance of concerts where amplified sounds are enough to cause noise induced hearing loss . RecogniTo balance the evidence, some studies have concluded from their data that there is no clear link between recreational noise exposure and hearing loss. For example, one research group concluded that most young users of personal listening devices are at low risk for noise induced hearing loss . HoweverThere is a strongly held view  that noise exposure effects are cumulative. Thus, in the short term, the effects of noise overstimulation may not be obvious, but the accumulated effects of damaging episodes eventually lead to significant hearing deficits. An important point here concerns the redundancy of hair cells in the cochlea. There are many more sensory elements than we need, and so considerable cell loss can occur before there are clinical signs of a problem. However, with repeated insults, our fixed complement of hair cells eventually runs out. This is one reason why noise induced damage in early years may not be immediately manifested but may become a problem in later life.For most healthcare professionals, hearing loss is largely described by the results of clinical tests such as the audiogram or speech threshold measures. In infants, hearing thresholds can be assessed objectively using auditory evoked potentials or otoacoustic emissions.Typically a deficit will be described as a hearing threshold loss in decibels (dB). It is important to realize that such basic threshold loss measures do not fully reflect a hearing “problem.” It is common to have a child with relatively good hearing thresholds but with significant problems in understanding speech. The ability to understand complex sounds can be reduced before pure tone audiometric threshold shifts are clinically significant. In the above review of the cochlear changes that accompany noise induced hearing loss, it was noted that deterioration in cochlear frequency analysis always accompanies the threshold elevation and is an important part of the overall deterioration in hearing function. In assessing possible noise induced hearing loss in children, clinicians might want to carry out or recommend a comprehensive hearing evaluation including speech discrimination tests.Beyond clinical tests results, there are broader ways of looking at hearing disability. The World Health Organization (WHO) has a scheme for assessing and describing hearing problems. This model distinguishes (1) impairment, (2) disability, and (3) handicap. Impairment is the actual loss of sensory function such as quantified by the clinical tests mentioned above. Disability is the “activity limitation” of an individual that results from the impairment . Handicap is a measure of “participation restriction,” that is, activities that a child may not be able to do because of the hearing problem. This might include making friends, keeping up at school, or being excluded from training for a certain career. For a child with noise induced hearing loss, the degree of deficit is likely to be mild or moderate, as opposed to severe or profound. However, such a loss might still be a barrier to effective communication, especially in noisy environments, such as the school classroom. It should also be recognized that mild to moderate hearing losses may not be immediately apparent to a child (in the same way that many older persons do not recognize that they have age-related hearing loss). Parents and clinicians should be vigilant and remember that measures of speech discrimination may more accurately reveal a hearing problem than simple (threshold) audiogram or hearing screening test.In a very young child, hearing problems can delay language development, and certainly if information is being missed at school, educational achievements can be reduced. For adolescents, communication difficulties can lead to social isolation, and there have been reports of suicide resulting from such situations. If hearing aid use is warranted, the adolescent may also have problems with the cosmetic appearance of the device or the stigma attached. The child may decide not to use the aid or choose to retreat to a small group or social isolation. In any case, it can be assumed that there will be quality of life implications. The quality of life impact may also be felt at a later age when job opportunities are restricted because of the hearing problem itself or a reduced educational attainment. The impact may well also be an economic one.The noise in our environment is no longer all “natural” and there numerous sources of amplified sounds that can damage hearing. In (western-based) industry, business, and the military, there is legislation or strict safety guidelines relating to noise exposure. In the areas of public entertainment  and personal entertainment devices, (MP3 or CD players and electronic games), regulations are not fully in place, and even if they were, it would be difficult to achieve compliance.Fortunately, there are numerous public awareness campaigns on the dangers of noise exposure, and there are some educational programs in schools that teach children that hearing loss can result from listening to loud sounds . Much useful information is now available online. A very rich and informative website is WISE EARS  launchedThere are numerous websites offering advice and promoting awareness. For example, KEEP IT HEAR  is “A NoNote on the table of various levels of example sounds-there is also an indication of how long an exposure to that sound can be considered safe. This is an important concept with regard to noise induced hearing loss. It is not just the sound intensity but also the duration of the exposure that determines its potential to cause cochlear damage. The efficacy of this program in changing behavior has been proven effective in a validation study .In recent years, the manufacturers and distributors of personal entertainment devices have been providing (in package) warnings and practical advice relating to the risks of noise induced hearing loss. It would be good to suppose that this was good corporate responsibility, as opposed to just being a hedge against possible litigation. In any case, such instructions, if attended to, can help prevent noise induced hearing loss.Many parents (and children) have concerns about the levels of sound exposure from personal entertainment devices and the risks of hearing loss. One common problem is a tendency to turn up the device volume to overcome the surrounding noise, which itself can be substantial. It is useful to suggest that adjustment of the device volume is made in a relatively quiet environment, to a level that is comfortable and then avoid increasing the volume further even in noisy settings. Our general auditory experience can tell us what is comfortable and what is too loud; perhaps parents should help younger children find that level. Some other useful advice is to get a child into the habit of checking if others nearby can also hear the music. If so it may be set too loud, although this of course will depend on the type of earphone in use. Mention was made above of the notion that it is not just the intensity of noise that is a problem, but also the duration of exposure. In this regard, for a young person who is constantly listening to music, it is advisable to take periodic 15–20 minute breaks, to allow the inner ear to “recover.”Another issue relates to the type of earphone or headphone used. The least risky in terms of the potential to do damage are loose fit earbuds that do not insert tightly into the ear canal. They are typically small transducers and do not output acoustic energy directly into the confined space of the ear canal. On the other hand, the listener is not insulated from the environmental noise and thus there is often a tendency to increase volume accordingly. Perhaps for the “careless” child, this type of earphone is the best. If the child is more responsible, then a type of earphone that blocks the outside noise can be recommended. This can provide the ear with a better sound  and obviate the need to increase volume to compete with environmental noise. These can be earbuds that fit snugly right into the ear canal or a larger headphone that fits against or around the ear. The downside for these transducers is that they can actually produce very intense signals either because sound energy is transmitted into a closed space or because of the size of the transducer diaphragm in the case of large headphones. For the serious music lover, active noise- reduction earphones are a nice luxury, but they are not very practical for children and may isolate an individual too much from the outside world.This paper started by describing the cochlear hair cell damage that can result from acoustic trauma and has emphasized the fact that there is little recovery and no regeneration of damaged hair cells. In this sense, there is really no treatment for noise induced hearing loss other than hearing aids that cannot fully restore normal hearing. With this being the case, all healthcare professionals should pay considerable attention to the education of parents and children about hearing loss prevention.


Objective. To describe a case of acute zonal occult outer retinopathy (AZOOR) in an active duty patient. Methods. In this paper we studied fundus photographs, optical coherence tomograph, Humphrey visual field 30-2, fundus autofluorescence images, fluorescein angiograms, and electroretinography. Results. Exam findings on presentation: a 34-year-old American Indian female presented with bilateral photopsias, early RPE irregularity, and an early temporal visual field defect. Progression RPE damage and visual field defect along with ERG findings support final diagnosis of AZOOR. Conclusion. AZOOR may initially be identified as a broader category of disease called the “AZOOR complex of disorders”. Specific visual field defects, ERG results, and clinical exam findings will help distinguish AZOOR from other similar disorders. Acute zonal occult outer retinopathy (AZOOR) was first described by Gass who noted in these patients a loss of outer retinal function, minimal fundus changes, irreversible ERG abnormalities, and permanent visual field loss . Other cA 33-year-old active duty American Indian female initially presented with bilateral photopsias and temporal visual field defects. She was referred from a community ophthalmologist with the diagnosis of a “possible white dot syndrome”. She has a history of high myopia (−6.75D OD/−7.00D OS).Her BCVA was 20/20 OD and 20/30 OS. The anterior segment ophthalmoscopic findings were normal bilaterally. Posterior segment evaluation showed that no vitritis, scalloped and circular RPE defects OU, and a known previous chorioretinal scar OS Figures  and 2. Humphrey visual field 30-2 revealed enlarged blind spots and temporal scotomas of both eyes Figures . The temAZOOR is a rare eye disease, marked by typical characteristics of photopsias, visual field defects, abnormal ERG, and subtle changes to the fundus. It is often found in young myopic females, similar to other white dot syndromes of MEWDS and PIC , 5. It wDespite the focal nature of the AZOOR clinically, there is electrophysiological evidence of global and generalized dysfunction affecting both the cone system and the retinal pigment epithelium . Such a The visual field pattern loss in AZOOR is most frequently temporal and complete. Initially, the visual field loss is progressive and then it becomes persistent . OccasioThe etiology of AZOOR remains unknown but a viral versus an inflammatory cause is suspected. The global electrophysiological abnormality may favor an inflammatory etiology over a viral cause . Viral-mNo treatment has been shown to effectively treat AZOOR. While one-third of patients may develop recurrence and portend a poorer visual outcome, the majority of patients have one episode with good visual recovery. According to Gass' series of patients 88% retain vision of 20/40 or better once the disease has subsided. In Gass' same patient population, improvement of the patient's vision occurred 6 months from initial presentation , 9.


Health care-associated infections (HCAI) result essentially from cross-transmission of pathogenic microorganisms by the hands of healthcare workers (HCW). Their care represents a universal challenge in practice.Our study aimed to measure HCW compliance with hand hygiene.We conducted a direct observance of hand hygiene compliance of HCW over a period of three months, based on the WHO’s “five indications of the hand hygiene” approach.For a total of 338 opportunities, the rate of global observance of hand hygiene was 36.1% with 80.3% of handrubbing realized. According to the department, this rate of observance was variable: Pneumology (42.3%), Thoracic Surgery and Cardiovascular (58.6%), Neurology (20%), Neurosurgery (24%), Emergency (25%), Laboratories (30%), Infectious diseases (39%), Psychiatry (33.3%), ORL (25%), Oral department (44.4%). According to the professional category, the observance was the following one: doctors (50.6%), nurses (34%) auxiliaries (29.1%) other nursing staffs (43.8%). The level of use of Alcohol-based handrub (ABHR) during hand hygiene was: auxiliaries (93%), doctors (82.1%), nurses (75.8%), others (14.3%). The observance of ABHR according to "five indications" was 87.7% before patient contact, of 83.3% before aseptic procedure, 44.4% after a risk of body fluid exposure, of 78% after patient contact and of 100% to the immediate surroundings of patient.Observance of hand hygiene with ABHR is still low in the structure. A training program coupled with a sharing experience of outcomes of the survey should allow to improve it.None declared