Identification of the graphlets that a vertex in an explored graph touches can provide useful information about the local structure of the graph around that vertex. Actually finding all graphlets in a large graph can be time-consuming, however. As the graphlets grow in size, more different graphlets emerge and the time needed to find each graphlet also scales up. If it is not needed to find each instance of each graphlet, but knowing the number of graphlets touching each node of the graph suffices, the problem is less hard. Previous research shows a way to simplify counting the graphlets: instead of looking for the graphlets needed, smaller graphlets are searched, as well as the number of common neighbors of vertices. Solving a system of equations then gives the number of times a vertex is part of each graphlet of the desired size. However, until now, equations only exist to count graphlets with 4 or 5 nodes. In this paper, two new techniques are presented. The first allows to generate the equations needed in an automatic way. This eliminates the tedious work needed to do so manually each time an extra node is added to the graphlets. The technique is independent on the number of nodes in the graphlets and can thus be used to count larger graphlets than previously possible. The second technique gives all graphlets a unique ordering which is easily extended to name graphlets of any size. Both techniques were used to generate equations to count graphlets with 4, 5 and 6 vertices, which extends all previous results. Code can be found at  A multitude of domains use graphs as a modeling tool. Obvious uses include the modeling of networks, such as social, communications and transport networks. Plenty of metrics exist to characterize them, for instance lengths of shortest paths or size of clusters of vertices. In recent years, though, graphlets are gaining more popularity as a method of characterizing graphs.G consists of a set of vertices, called V, and a set of edges E, so that each edge connects two vertices. As such, an edge can be notated by listing the couple of vertices it connects:order, the number of edges its size. Isomorphisms between two graphs are bijections which map one graph’s vertices to the other’s so that both edge sets are the same. The set of isomorphisms between two graphs G and H is given byG ≃ H ⇔ Iso≠∅. Isomorphisms from a graph to itself are called automorphisms:The following symbols will be used throughout this paper for clarity. A graph th graphlet in this numbering will be called graphlet x, all graphlets of order n are called n-graphlets. An induced subgraph of a graph is defined as a subgraph containing a collection of vertices and all edges between those vertices. A graph H is an induced subgraph of graph G =  ifexplored graph. Specific induced subgraphs can be searched within an explored graph, either listing each occurrence of the subgraph or simply counting them. When these induced subgraphs of the explored graph are isomorphic to a graphlet, all vertices in such an induced subgraph are said to touch that graphlet. The induced subgraph itself is then also said to be an instance of that graphlet.Pržulj, Corneil and Jurisica define graphlets in  as connex is given byorbit n and vertices of an explored graph touching a graphlet are also said to touch the orbit. The graphlet degree distribution (GDD) for a specific orbit o and a certain number k is defined as the number of vertices in the explored graph that touch orbit o exactly k times  == null then       equations[e1.lowestOrbit] := e1else if equations[e1.lowestOrbit].graphlets == e1.graphlets then   merge    equations[e1.lowestOrbit] := end if   end for  end for return equations end functionThe presented method can generate equations for graphlets of any order. However, the method used to find a linearly independent set, as well as the actual interpretation of the equations, need an actual naming scheme for the generated orbits. Like done before, we will give each graphlet and orbit a number. Unlike the graphlet numbering used before, however, we will try to generate the ordering of the graphlets automatically. This way, the numbering can be extended to larger graphlets without the need to change the code.The criteria for selection of an independent system of equations impose a partial ordering on the orbits’ numbers. If one orbit can be created by adding an edge to another, the first must have a higher number than the second. Orbits that cannot be created from each other by only adding or only removing edges do not need a particular ordering. To simplify matters, first an ordering for graphlets will be made, then the orbits within each graphlet will be ordered.K3,3 (panel B) does not. As such, they cannot be discriminated on basis of number of edges, degree of vertices, orbits or which type of vertices are connected. One can imagine this kind of problem will only get worse with increasing order.Choosing to apply intuitive rules, that can be easily checked at first sight by an observer, translates poorly to larger graphlets. For example, both 6-graphlets in n ∗ n matrix. The value at row r and column c is 1 if there is an edge between vertex r and c and 0 if there is not. In To avoid such problems, graphlets are represented in triangular matrix form. In matrix form, an n-graphlet is represented as an If such a string is made for each graphlet of a certain order, and those strings are sorted lexicographically, a unique ordering for the graphlets is established. Any added edge will change a 0 into a 1, which will make the string lexicographically larger, and the new graphlet’s number will therefore be larger than the old one’s. This means that using this ordering to number the graphlets gives rise to a numbering following the partial ordering that was required.In practice, the ordering was made by generating all possible string forms of graphlets of a certain size in order with a binary counter. These are then converted to actual graphlets and added to an ordered set so they keep their ordering but duplicates are not allowed.The orbits are numbered in the order that their vertices first appear in the lexicographically smallest string representation. Vertex 0 in this representation will always be in the first orbit, vertex 1 can either be in the same orbit or start orbit 2, and so on.p is the number of vertices that form 3-vertex paths starting with a and b, i.e. all neighbors of b but not a, except a itself. This equation is the corresponding equation that appears in [The equations generated for 4-graphlets can be found in pears in .Similarly, the equations for 5-graphlets, which can be found in The new equations for 6-graphlets are shown in As is expected, most of the graphlets up to order 5 have a different naming here than in Pržulj’s scheme. This is shown in For ease of comparison between the automatically generated equations and the equations in , the oriAll graphlets and orbits of order 6 were identified. A total of 112 graphlets and 407 orbits were identified and can be found in https://github.com/IneMelckenbeeck/equation-generator and https://github.com/IneMelckenbeeck/graphlet-naming.A new algorithm has been developed to automatically generate equations that facilitate counting the number of times each node of an explored graph touches each orbit of graphlets of a certain order. This algorithm can create an independent set of equations to calculate graphlet degree distributions without restriction of the order of the graphlets. In addition, a new algorithm to automatically name graphlets and orbits has been created, which enables the use of higher-order graphlets. Both algorithms were programmed in Java, the source code is available at All 6-graphlets were ordered and named by this algorithm, and the equations that enable efficient counting of their orbits were generated. This is a large step towards using graphlets with an order of 6 and larger.The algorithms presented in this paper enable the use of an ORCA-like counter for graphlets of any size. However, the ORCA code itself is highly optimized for counting 4- and 5-graphlets, making it impossible to use the equations for any other order with the existing code. The next step is thus to develop an efficient and order-independent algorithm that makes efficient use of these equations to actually count the times each vertex in an explored graph touches each orbit.S1 Equations(PDF)Click here for additional data file.S2 Equations(PDF)Click here for additional data file.S3 Equations(PDF)Click here for additional data file.S1 FigAll graphlets of order 6 are shown in the newly introduced order. In the lower left corner of each page, the graphlet’s orbits are listed in their order.(PDF)Click here for additional data file.


Alternaria sp. MG1, an endophytic fungus previously isolated from Merlot grape, produces resveratrol from glucose, showing similar metabolic flux to the phenylpropanoid biosynthesis pathway, currently found solely in plants. In order to identify the resveratrol biosynthesis pathway in this strain at the gene level, de novo transcriptome sequencing was conducted using Illumina paired-end sequencing. A total of 22,954,434 high-quality reads were assembled into contigs and 18,570 unigenes were identified. Among these unigenes, 14,153 were annotated in the NCBI non-redundant protein database and 5341 were annotated in the Swiss-Prot database. After KEGG mapping, 2701 unigenes were mapped onto 115 pathways. Eighty-four unigenes were annotated in major pathways from glucose to resveratrol, coding 20 enzymes for glycolysis, 10 for phenylalanine biosynthesis, 4 for phenylpropanoid biosynthesis, and 4 for stilbenoid biosynthesis. Chalcone synthase was identified for resveratrol biosynthesis in this strain, due to the absence of stilbene synthase. All the identified enzymes indicated a reasonable biosynthesis pathway from glucose to resveratrol via glycolysis, phenylalanine biosynthesis, phenylpropanoid biosynthesis, and stilbenoid pathways. These results provide essential evidence for the occurrence of resveratrol biosynthesis in Alternaria sp. MG1 at the gene level, facilitating further elucidation of the molecular mechanisms involved in this strain's secondary metabolism. Escherichia coli or yeast strains through genetic modification . Although, these key enzymes were identified in microorganisms separately, the complete biosynthesis pathway has not been predicted in any strain. We previously found Alternaria sp. MG1, an endophytic fungus isolated from Merlot grape, could produce resveratrol in vitro , a strain previously isolated from the cob of Merlot grape, was used in the study and stored in the China Center for Type Culture Collection , 20 g glucose, 1000 mL water]. The cultivation was carried out at 28⋅C and 120 rpm in a rotary shaker. After a cultivation time of 21 h, the optimum period previously found to produce abundant resveratrol . After washing twice with sterile water, the collected cells were immediately stored in liquid nitrogen until further analysis. Extraction of the total RNA from the cells was performed using a Spin Column Fungal total RNA Purification Kit . RNA extraction quality and quantity was analyzed using a NanoDrop 2000 Spectrophotometer  gel electrophoresis, and an Agilent 2100 Bioanalyzer .After total RNA extraction, the RNA-seq library was constructed according to the previously reported method  technology, the quality reads were assembled using the Illumina HiSeq 2500 platform. The quality score of the raw reads was set at Q30 or above. Generally, the raw reads contain tiny minority primer sequences, adapter sequences, and other potential contaminants. Prior to subsequent analysis, the clean reads were filtered from the raw reads by removing the reads with only adaptor and unknown nucleotides. Data analysis and base calling were performed using Illumina instrument software. Raw sequence data were subsequently deposited in the NCBI database with the SRA study accession number, http://bowtie-bio.sourceforge.net/bowtie2/manual.shtml) and normalized into RPKM (Reads per kb per million reads) values according to formula (1).To evaluate the expression level of all reads, each unigene was compared with the unigene database by using Bowtie  annotations and the distribution of gene functions for each unigene using a cut-off value of 10−5 , Swiss-Prot, TrEMBL, and Cluster of Orthologous Groups of proteins (COG) using BLAXTX, and against the NCBI nucleotide database (Nt) using BLASTN, with a cut-off E-value of 10Alternaria sp. MG1 were translated from a nucleotide sequence using a web translate toolTo align the sequences and construct the phylogenetic tree, the chalcone synthase amino acid sequences of Alternaria sp. MG1, a mixed cDNA sample representing the high resveratrol-producing phase of this strain was prepared and sequenced. Each sequenced sample yielded 2 × 100 bp independent reads from either end of a cDNA fragment. After stringent quality assessment and data filtering, 22,954,434 reads (4.64 Gb) with 53.73% (GC%) and 89.42% Q30 bases  were selected as high quality reads for further analysis  with the comparison results, the gene expression level was estimated. Here, the RPKM values were used to represent the expression abundance of each unigene. The distribution of each gene's expression is shown in Figure −5. The obtained overall functional annotation is depicted in Table Several complementary approaches were also used to annotate the assembled sequences, including aligning the sequences with those deposited in diverse databases such as Nr, Nt, Swiss-Prot, COG, GO, and KEGG. The best alignment was selected from matches with an E-value of < 10The Gene Ontology (GO) project is a collaborative effort to address the need for consistent descriptions of gene products across databases. A further functional classification of all unigenes was performed using a set of plant-specific GO slims Figure . A totalTo evaluate the completeness of our transcriptome library and the effectiveness of our annotation process further, we used the annotated unigene sequences to search for the genes in the COG classifications. All unigenes were subjected to a search against the COG database for functional prediction and classification. Overall, 4954 sequences showing a hit with the Nr database were successfully assigned to COG classifications  were “N-glycan biosynthesis” (41 unigenes) and “various types of N-glycan biosynthesis” (11 unigenes), “glycosaminoglycan degradation” (2 unigenes), “glycosylphosphatidylinositol (GPI)-anchor biosynthesis” (21 unigenes), and “glycosphingolipid biosynthesis” (11 unigenes), and “other glycan degradation” (9 unigenes). The annotated pathways involved in terpenoid and polyketide metabolism (35 unigenes) were “terpenoid backbone biosynthesis” (14 unigenes), “limonene and pinene degradation” (11 unigenes), “diterpenoid biosynthesis” (2 unigenes), and “carotenoid biosynthesis” (8 unigenes). Metabolism pathways for the biosynthesis of secondary metabolites (58 unigenes) were also annotated as “phenylpropanoid biosynthesis” (23 unigenes), “flavonoid biosynthesis” (7 unigenes), “flavone and flavomol biosynthesis” (3 unigenes), “stilbenoid, diarylheptanoid, and gingerol biosynthesis” (3 unigenes), “isoquinoline alkaloid biosynthesis” (10 unigenes), “caffeine metabolism” (4 unigenes), and “glucosinolate biosynthesis” (1 unigene).Alternaria sp. MG1, all unigenes involved in resveratrol biosynthesis were specifically selected in the study. As expected, many genes involved in the pathway were successfully annotated in COG and KEGG databases. A total of 84 unigenes were annotated in the four major metabolism pathways leading to the formation of resveratrol, including glycolysis (ko00010), phenylalanine biosynthesis (ko00400), phenylpropanoid biosynthesis (ko00940), and stilbenoid biosynthesis (ko00945). The annotated unigenes and corresponding RPKM values are indicated in Figure In order to reveal the genetic basis of the resveratrol biosynthesis pathway in is ko0094. The annGlycolysis (ko00010) contributes to the early stage of resveratrol biosynthesis by converting different substrates  to phosphoenolpyruvic acid  encoding four enzymes were annotated in the two pathways for converting L-phenylalanine into resveratrol Figure . ImportaAegilops tauschii stilbene synthase (EMT02178.1) and A. tauschii chalcone synthase (EMT09075.1) than other enzymes, while c4454 was similar to Alternaria alternata polyketide synthase (JX103640) and Pyrenophora tritici-repentis chalcone synthase (XM001934899).Chalcone synthase was reported as an isoenzyme of resveratrol synthase having low activity in resveratrol biosynthesis  and Oryza sativa chalcone synthase (54%). The sequence homology of c8532 with selected proteins was 42% for Persicaria minor, 45% for Vitis vinifera, 43% for Arachis hypogaea, and 42% for Polygonum cuspidatum. c4544 had the highest homology with Alternaria alternate polyketide synthase (96%), and less homology with P. tritici-repentis (78%), Neurospora crassa (49%), Colletotrichum graminicola (47%), and Aspergillus oryzae (45%)  Figure . The tworepentis 8%, NeuroAs a newly explored biological resource, endophytic fungi offer an excellent opportunity to produce active compounds without the same limitations as plant resources. However, critical obstacles such as low and unstable production of metabolites greatly inhibit the application of these fungi  and Trichosporon asahii (XM014326038.1); 4CL in Rasamsonia emersonii (XM013469671.1) and Colletotrichum gloeosporioides (XM007280559.1); and CHS/ST in Marssonina brunnea (XM007294196.1) and Aspergillus flavus (XM002380742.1). However, no complete pathway for microorganisms has been predicted. In this study, a total of 115 pathways represented by a total of 2701 unigenes were predicted in Alternaria sp. MG1. The predicted metabolic pathways in this strain contributing to resveratrol biosynthesis were that of glycolysis/gluconeogenesis (ko00010), phenylalanine, tyrosine, and tryptophan biosynthesis (ko00400), phenylpropanoid biosynthesis (ko00940), and stilbenoid, diarylheptanoid, and gingerol biosynthesis (ko00945). Overall, 20 unigenes encoding four enzymes were annotated in the resveratrol biosynthesis pathway. This work not only presented the first de novo transcriptome sequencing analysis of RNA from Alternaria sp. MG1, but also revealed the possible candidate genes of relative pathways in this fungus for the first time. Two unigenes were successfully located in a cluster of chalcone synthase and stilbene synthase by phylogenetic analysis, with high sequence homology within the CHS-like domain of active and product binding sites. In addition, we obtained abundant transcriptomic data for Alternaria sp. MG1, and carried out an integrated analysis on the gene expression of resveratrol biosynthesis. These results can be used to discover the genetic foundation of Alternaria sp. MG1, especially the resveratrol biosynthetic pathway, as well as the synthesis pathways of other active compounds. This understanding could pave the way for genetically improved varieties of Alternaria sp. MG1 with increased secondary metabolite yields.So far, three  of the four key enzymes have been identified in minority microorganisms respectively. There was little published regarding these key enzymes, but some genes have been recorded in the genebank. For example, PAL in More importantly, the putative transcriptome information obtained in this study will provide a significant contribution toward understanding resveratrol metabolism and biosynthesis and may help in facilitating elucidation of secondary metabolite molecular mechanisms in other microorganisms.This article does not contain any studies with human participants or animals performed by any of the authors.JC, the author of this paper was in charge of this study, including the experimental design and implementation, drafting the article, etc. JS, the corresponding author of this paper was supervising the Work. ZG, a member of this team. Assist to references collection, data analysis, article proofreading, etc. YZ, a member of this team. Assist to complete the experiment, data analysis, article proofreading, etc.This project was supported by the National Science-technology Support Plan Projects (No. 2015BAD16B02), the National Natural Science Fund (Grant No. 31471718), the Agriculture Department of China (Grant No. CARS-30), and the Northwestern Polytechnical University .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.


Manufacturing sector accounts for 18.1% of the total employed persons in Malaysia, which is known for the utilisation of noisy machinery to establish output. Even though it is compulsory for employers to notify any hearing loss cases to the authority annually, there is still scarcity of occupational hearing loss cases. Therefore, this study aimed to estimate the total number of workers exposed to occupational noise among workers in manufacturing industries in Malaysia.A field survey was carried out at 26 selected industries in five states Johor, Selangor, Pulau Pinang, Terengganu and Pahang that recorded highest noise induced hearing loss (NIHL) cases in 2011. Safety and Health Practitioners in each industry were interviewed and elicited information on the industrial classification, occupational noise management and level of exposure to workers. Noise exposures were categorised from noise exposure monitoring report. The percentage of high risk workers in each class was derived to estimate the number of exposed workers based on the Economic Census.Levels of occupational noise exposure among industries were 28% for 91-140 dBA and 72% for 86-90 dBA; which classified that all workforces were at high risk. Occupational noise-exposed workers were observed to be highest in the metal industry (2091) followed by textile (631) and food (439). The percentage of workers exposed ranged from 13.6% to as high as 68.9% in each industry. In addition 103,673 (39%) from total employment of 267,964 were estimated to be workers exposed to high risk noise. Although, employers provide awareness programme and hearing protection device but none of the industries implemented attenuation of it. A few industries showed inconsistency in annual audiometric test.This study indicates that the majority of workforce in manufacturing industry was exposed to high risk occupational noise and effective hearing conservation programme needs to be implemented for preventive measure.


Massive cellular insults lead to necrosis. It appears, however, that cells have some choice in the mode of their demise depending on the severity of the insult. Hypoxic cores in solid tumors2 or perfusion-impaired brain tissue in stroke,3 for instance are typically necrotic, but possess an apoptotic shell.Cell death can take many shapes. Programmed ‘apoptotic' and uncontrolled ‘necrotic' cell death mark the extremes of the spectrum of possibilities.6 Lysosomes can rupture during ischemic or traumatic cell injury,7 giving rise to both apoptotic and necrotic outcomes.The same can be seen for lysosomal damage.8 dedicated to the degradation of both intracellular material, such as damaged or old organelles, and extracellular components such as matrix proteins. The release of the lysosomal proteases into the cytosol sentences the cell to death by autodigestion.Lysosomes are cellular organelles with a proteolytic function and possess an impressive set of protease enzymesCell Death Discovery article,9 we looked at the fate of the major regulators of apoptosis, the bcl-2 protein family, in order to understand how cell fate upon lysosomal rupture is directed toward necrosis or apoptosis. This protein family contains both pro- and anti-apoptotic members and has been shown to be part of the proteolytic spectrum of cathepsin lysosomal proteases.10 We reasoned that a regulated degradation of these proteins, by affecting the equilibrium of pro- and anti-apoptotic signaling, might offer a means for switching between cell death forms.In a recent To this end, we imaged the proteolytic degradation of the major bcl-2 family representatives bcl-xl, bid and bax upon induced lysosomal disruption in real time using förster resonance energy transfer (FRET) microscopy of single cells. We created proteolytic FRET sensors by sandwiching full-length bcl-2 proteins between cyan and yellow fluorescent proteins. A strong FRET signal is detected in the intact sensor, which is lost upon cleavage , left.The bcl-2 sensors show a common sigmoidal ‘delay-snap' cleavage behavior: after a delay of tens of minutes, cleavage occurs rapidly and is completed within minutes. Among the observed bcl-2 members, anti-apoptotic bcl-xl is degraded before pro-apoptotic bid and bax. Cells thus appear to accumulate pro-apoptotic reactants in the early stages of lysosomal rupture.11 Our work places the bid shunt very early in lysosomal cell death signaling and this is the first time this event is caught ‘on film'.The pro-apoptotic bid showed a conspicuous behavior. Before the overall sigmoidal degradation, a pronounced and very rapid cleavage was observed in the first minutes of lysosomal lysis. Fast proteolytic processing of bid is used in apoptosis and might serve the same role in lysosomal lysis. In apoptosis, the caspase 8 protease activates bid by its truncation, directly connecting the extrinsic and intrinsic apoptotic signaling pathways. This mechanism is called the ‘bid shunt' and processing of bid by cathepsin B has been suggested to operate similarly.In order to gain a better understanding of the regulation of bcl-2 protein levels by cathepsins and the involvement in programmatic steps in necrosis and apoptosis, we investigated the interplay of both systems in more detail.Given the relatively low amount of lysosomes that were disrupted in the early stages after lysosomal disruption, we selected the neutral-active cathepsin B as likely candidate for most of the early proteolytic actions. Hence, we repeated the experiments in the presence of a selective cathepsin B inhibitor and an inhibitor for the thiol-cathepsin class, to which cathepsin B belongs. Inhibition of the thiol-cathepsins confirmed their predominant role in the degradation of bcl-2 family proteins, as the proteolysis of all three bcl-2 constructs was inhibited. Selective cathepsin B inhibition helped uncover its role in steering cell death. We found that early bid truncation was abolished, confirming cathepsin B as the responsible protease. Furthermore, when we limited lysosomal protease action to the time window of the early bid truncation, we detected late-apoptotic caspase 3/7 activation, showing that cells had chosen an apoptotic exit.Unexpectedly and paradoxically, bax and also bid exhibited a new accelerated proteolytic behavior upon selective inhibition of cathepsin B. The delay phase before the onset of cleavage was completely abolished, in particular for bax. As a general inhibition of the thiol-cathepsin classes resulted in the complete inhibition of the proteolysis of these same sensors, we concluded that their accelerated degradation upon cathepsin B inhibition is most likely caused by another, unidentified, thiol-cathepsin. This proteolytic cascade prevents the degradation of apoptosis-supporting proteins, shifting the balance toward apotosis. The identity of this cathepsin requires further research.Cells thus appear to be able to pull an ‘emergency brake' upon lethal injury with damaging of lysosomes. Starting with cathepsin B, an early programmatic protease cascade is initiated that appears to avoid necrosis in favor of apoptosis. In this apoptotic exit effort, cathepsin B preferentially degrades anti-apoptotic bcl-xl, activates bid by its rapid and selective truncation, and degrades a thiol-cathepsin to avoid the removal of apoptotic bid and bax , right.


Aims. To test the hypothesis that primed PMNLs in blood of chronic kidney disease patients release the active form of elastase and cathepsin G causing degradation of vital proteins and promote tissue damage.  Methods. RT-PCR, immunocytochemical staining, immunoblotting, and FACS analyses were used to study these enzymes in hemodialysis patients (HD) versus healthy normal controls (NC). Using PMNLs and endothelial cells cocultivation system we measure the effect of HD PMNLs on the endothelial VE-cadherin, an essential protein for maintaining endothelial integrity.  Results. Levels of elastase and cathepsin G were reduced in PMNLs of HD patients, while mRNA enzymes levels were not different. Elevated levels of the active form of these enzymes were found in blood of HD patients compared to NC.HD plasma had higher levels of soluble VE-cadherin present in three molecular forms: whole 140 kDa molecule and two fragments of 100 and 40 kDa. Cocultivation studies showed that primed PMNLs cleave the endothelial cadherin, resulting in a 100 kDa fragment.  Conclusions. Elastase and cathepsin G are elevated in the plasma of HD patients, originating from primed PMNLs. In these patients, chronic elevation of these enzymes contributes to cleavage of VE-cadherin and possible disruption of endothelial integrity. Storn plasma , 11. ThePMNL serine proteinases have been implicated in the pathogenesis of diseases associated with PMNL priming such as rheumatoid arthritis , 13. PMNIn this study, we sought out to test the hypothesis that even before dialysis HD PMNLs are primed, thus contributing to elevated levels of elastase and cathepsin G in the plasma. We have also evaluated cleavage of VE-cadherin mediated by these proteinases found in HD plasma.Our findings consist of elevated levels of these proteins in HD plasma. Plasma of HD had higher levels of VE-cadherin fragments, the result of this molecule cleavage by active elastase and cathepsin G. HD PMNLs as well as purified enzymes cleaved VE-cadherin from cultured endothelial cells. Taken together, this study provides a novel mechanism by which active elastase and cathepsin G originating from primed PMNLs of HD patients can initiate endothelial dysfunction.Ten hemodialysis patients and healthy control subjects were enrolled in this study . Blood f7 cells per isolation) were resuspended and counted in phosphate buffered saline  containing 0.1% glucose. PMNL priming was assessed by the rate of superoxide release [−7 M phorbol 12-myristate 13-acetate . The assay is based upon superoxide dismutase (SOD) inhibitable reduction of 80 μM cytochrome C  to its ferrous form. The change in optical density was monitored at 549 nm, as described previously [μL of blood was incubated for 10 min with anti CD11b-PE conjugated monoclonal antibody , followed by RBC lysis . To enable gating on the PMNL population, anti CD16, conjugated to PC5 monoclonal antibody , was used. Surface levels of CD11b on PMNLs are expressed as mean fluorescence intensity (MFI), after subtracting the nonspecific background.PMNLs were isolated as described previously . Isolate release , and by  release . The rateviously . ExpressμL aliquots of RT reaction incubated with 0.2 mM dNTPs, 2 μL of Taq DNA polymerase buffer, 0.6 U of TaqDNA polymerase , and 10 pmoles of the specific primers. The oligonucleotide sequence for elastase, according to Hirata et al. [μL) of the amplified cDNA of elastase and cathepsin G were separated on 1% agarose gel electrophoresis, visualized by ethidium bromide staining, and compared to a housekeeping gene, β-actin. The 200 bp product of actin was amplified using the following primers: sense (5′-CCTTCCTGGGCATGGAGTCCTG-3′), and antisense (5′-GAGCAATGATCTTGATCTTC-3′). The densities of the enzyme PCR products were calculated in each gel relative to the actin product.RNA was isolated using Tri Reagent according to the manufacturer's (Sigma) instructions. Complementary DNA was synthesized according to Krug and Berger , using aa et al. , is sensa et al. , is sens(1) Intracellular Levels of Elastase and Cathepsin G in PMNLs. The levels of intracellular elastase and cathepsin G were measured in whole blood by flow cytometry using polyclonal rabbit anti-human elastase and anti-human cathepsin G antibodies , followed by a secondary FITC-conjugated fluorescent anti-rabbit antibody . PMNLs were permeabilized with the FIX & PERM cell permeabilization kit. Anti CD16- PC5  was used for gating on PMNLs. Total rabbit IgG antibodies , followed by a secondary FITC-conjugated fluorescent anti-rabbit antibody, served as nonspecific controls of the fluorochrome. The results are presented as MFI per cell after subtracting the nonspecific background.(2) Localization of Elastase and Cathepsin G in PMNLs. Human PMNLs were stained by indirect immunohistochemical staining. Whole blood slides were prepared by fixation and permeabilization with methanol . The slides were stained with specific anti-elastase and anti-cathepsin G antibodies . The Histofine Simple Stain kit  was used for immunohistochemical staining of PMNLs according to the manufacturer's instructions.β-mercaptoethanol, the plasma was boiled at 100°C for 5 minutes. Samples were loaded onto 15% polyacrylamide SDS gels for separation of proteins and then transferred to nitrocellulose filters by semidry transfer . The filters were blocked by 1% fat milk for 1 hour at room temperature and then incubated at room temperature, first with rabbit anti-human elastase/rabbit anti-human cathepsin G antibodies   for 2 hours, followed by incubation with goat anti rabbit-HRP conjugate  for an additional hour. The amount of the enzyme in each sample was calculated according to a positive control loaded on the same gel, active elastase (Sigma-Aldrich-cat. number E-8140)/active cathepsin G .Plasma of NC subjects and HD patients was depleted of albumin and immunoglobulins by ProteoPrep Blue Albumin Depletion Kit (PROT-BA KIT) . Following the addition of Laemli loading buffer containing 3% The enzyme signal was detected on X-ray films using the chemiluminescence reagents of the EZ-ECL kit. The densities of the enzyme bands were determined by the BioCapt and Bio-Profile (Bio-1D) software.Human umbilical vein endothelial cells (HUVEC) were cultured as described by Jaffe et al. , with mi5 cells in six-well plates  for 2 days under subconfluent conditions. Experiments were performed always between passages 2 to 4. In all experiments, prior to the addition of PMNLs, HUVEC were deprived of culture medium and kept in phosphate buffered saline (PBS) for 90 min at 37°C [μm pore size cell culture inserts  were placed into each well on top of HUVEC in direct contact with the medium, 1 mL of PBS [6) from NC or HD were seeded on these inserts and coincubated for 15 minutes at 37°C with HUVEC, as previously described [HUVEC were seeded in growth medium at a density of 1 × 10 at 37°C . To prevL of PBS . PMNLs ((1) By Flow Cytometry. HUVEC, following 15 minute of cocultivation with PMNLs, were rinsed with PBS, scraped, centrifuged, resuspended, and incubated for 10 minutes with mouse anti VE-cadherin monoclonal antibody , followed by incubation with PE conjugated anti-mouse antibody . Irrelevant IgG-PE served as a nonspecific control of the fluorochrome . (2) By Immunohistochemical Staining. HUVEC were grown on a chamber, a procedure that does not require cell transfer prior to visualization/staining, and cocultivated with PMNLs as previously described [escribed . After c(1) In Plasma. Soluble VE-cadherin was measured using western blot analysis. The plasma of NC subjects and HD patients was depleted of albumin and immunoglobulins. Following the addition of Laemli loading buffer containing 3% β-mercaptoethanol, the plasma was boiled at 100°C for 5 minutes. Samples were loaded onto 6% polyacrylamide SDS gels as described above. The whole 140 kDa molecule and the 100 kDa fragment were detected using mouse anti-human VE-cadherin , while the 40 kDa fragment was detected using mouse anti-human VE-cadherin , which is not able to recognize the 140 kDa and the 100 kDa molecules. Thus, due to the different antibodies used, plasma sampled was loaded onto two gels, one stained for the evaluation of 100 + 140 kDa forms and one for the 40 kDa form. The levels of both whole and cleaved forms of VE-cadherin were assessed in HD plasma and compared with those of NC plasma. To calculate the levels of the enzymes, the bands were all compared to the same plasma sample run on all gels serving as a normalizing control.(2) In the Cocultivation Media. The media of four cocultivation experiments were pooled in order to get detectable amount of these proteins. These media samples were treated with Vivaspin concentrators  for improved recovery of the low-concentration protein samples. Following the addition of Laemli loading buffer containing 3% β-mercaptoethanol, the plasma was boiled at 100°C for 5 minutes. Samples were loaded onto 6% polyacrylamide SDS gels as described above using mouse anti-human VE-cadherin .P < 0.05.Data is expressed as mean ± SD. In the boxes and whiskers presentations, the horizontal line in the middle shows the median (50th percentile), the top and bottom of the box show the 75th and 25th percentiles, respectively, and the whiskers show the maximum and the minimum values. The nonparametric Mann-Whitney test was used for comparing two independent groups. The two-paired Wilcoxon Signed Ranks test was used for comparing two dependent groups. Statistical significance was considered at  6 cells/10 min, resp., P < 0.05). The membrane levels of CD11b were higher in PMNLs isolated from HD patients than in PMNLs from NC .We reported previously that PMNLs from HD patients are primed . To confP = ns; and 0.74 ± 0.1 versus 0.86 ± 0.47 relative density units, respectively, for cathepsin G; P = ns.Elastase and cathepsin G mRNA levels were not significantly different in NC versus HD PMNLs: 0.39 ± 0.14 versus 0.33 ± 0.22 relative density units, respectively, for elastase; P < 0.05 and 35 ± 5.2 versus 12.8 ± 1.4 MFI, resp., for cathepsin G; P < 0.05).Flow cytometry measurements of elastase and cathepsin G in PMNLs measured in whole blood Figures  showed hr = −0.43 and r = −0.51, resp.; P < 0.05). This negative correlation indicates that the higher the priming the lower the amount of the intracellular enzymes.A significant negative correlation was found between the fluorescent intensity of intracellular elastase and cathepsin G and membrane CD11b expression on PMNLs,  .Plasma of NC and HD was depleted of albumin and immunoglobulins in order to enrich it with elastase and cathepsin G. After depletion, plasma proteins were separated on SDS-PAGE followed by western blot analysis . HD plas Figures . However Figures  (r = −0.P < 0.05), and the cleaved form, 100 KDa   , suggesting that these proteins are involved in the degradation of VE-cadherin. No significant correlation was found between plasma active elastase and cathepsin G and the 140 and 40 KDa VE-cadherin forms.In addition, significant positive correlations were found between plasma active elastase and cathepsin G and the 100 KDa soluble VE-cadherin Figures  (r = 0.5P < 0.05). Cocultivation of HUVEC with HD PMNLs resulted in a greater increase in VE-cadherin staining .Control HUVEC and HUVEC exposed to NC PMNLs show light staining which indicates that the anti VE-cadherin did not bind to the VE-cadherin . HoweverWe could not detect any soluble VE-cadherin in the control HUVEC which was not exposed to PMNLs. On the other hand, there was a detectable amount of soluble 100 kDa VE-cadherin in the cocultivation media when HUVEC were exposed to PMNLs . When HUThe results of the present study demonstrate that the intracellular levels of elastase and cathepsin G in primed peripheral PMNLs of HD patients, before starting hemodialysis session, are significantly lower compared to healthy controls although their mRNA levels are similar. Lower intracellular levels of these proteases are associated with a significant increase in their plasma concentration, especially of the active form. This suggests that PMNL priming, common to HD patients, causes an increased release of these enzymes, resulting in their higher plasma levels in HD. Plasma from HD patients also contains higher levels of soluble VE-cadherin, in three molecular forms: 140 kDa, the whole molecule and two molecular fragments of this protein, 100 and 40 kDa.We investigated the degree of PMNL priming of HD patients and the possible correlation between PMNL priming and the release of elastase and cathepsin G. First we supported our previous studies showing that PMNLs in HD patients are primed by using two different markers: elevated levels of CD11b and higher rate of superoxide release. Next, using immunocytochemical staining, immunoblotting, and FACS analyses we demonstrated that the staining intensities of the intracellular elastase and cathepsin G are reduced in the primed PMNLs of HD patients versus NC. A significant negative correlation was found between the intensity of intracellular elastase and cathepsin G and the degree of PMNL priming, as assessed by membrane CD11b expression, suggesting that the release of these proteolytic enzymes is greater when PMNLs are primed. Moreover, our findings refute the possibility that lower synthesis of these enzymes in the HD PMNLs caused their reduced intracellular levels, since the transcription levels of these enzymes were comparable in HD and healthy controls. This is in accordance with previous studies demonstrating that azurophilic granule proteins, such as these serine proteinases, are synthesized only at the promyelocyte and metamyelocyte stages, and remain stored in granules throughout terminal granulocytic differentiation , 28.Since the intracellular protein levels of these enzymes were lower in HD, one would expect to find these enzymes in the surrounding milieu, the blood. As expected, significantly elevated levels of both proteolytic enzymes were found in blood of HD patients compared to NC.α1-antitrypsin (α1-AT) and α1-antichymotrypsin (α1-ACT) [The continuous release of these enzymes from PMNLs was already demonstrated during hemodialysis sessions , but to (α1-ACT) , 11. The in vivo [In the “response-to-injury” hypothesis suggested by Ross, atherosclerosis begins as a response to chronic minimal injury to the endothelium . This in in vivo . Soeki e in vivo . We implThis study demonstrates that HD plasma contains higher levels of soluble VE-cadherin present in three molecular forms: whole 140 kDa molecule and two fragments, the 100 and the 40 kDa forms. The 100 kDa protein is probably a product of the proteolysis of VE-cadherin by elastase and cathepsin G . This as in vitro experiments showed that HD PMNLs can mediate cleavage of VE-cadherin from endothelial cells. This cleavage resulted in intense staining of the 40 kDa fragment on endothelial cells concomitantly with the appearance of the 100 kDa in the culture media. These results apparently conflict with our findings regarding low levels of elastase and cathepsin G found in HD PMNLs compared to controls. Yet, we propose that while in NC PMNLs these enzymes are stored within specific granules, HD PMNLs secrete their enzyme content to the surrounding milieu. Therefore, even trace amounts could elicit VE-cadherin cleavage as describe herein. The potential role of elastase and cathepsin G released from primed HD PMNLs in mediating VE-cadherin cleavage is further supported by our in vitro experiments where applying purified elastase and cathepsin G to HUVEC resulted in an increase staining of the 40 kDa fragment.Our in vitro studies utilizing these proteinases on endothelial cells are in agreement with previous study demonstrating that after treatment with cathepsin G, endothelial cells became contracted and did not interact with neighbor cells. These effects depended on concentration and length of exposure to cathepsin G and ultimately cells became star-shaped until totally detached [Ourdetached .For conclusion, this study shows decreased elastase and cathepsin G expression in HD PMNLs while increased in HD plasma, most importantly in their active form. Furthermore, higher levels of soluble VE-cadherin were found in HD patient plasma. Thus, this study can provide a mechanism by which active elastase and cathepsin G are released from primed PMNLs, mediate VE-cadherin cleavage, and initiate endothelial dysfunction in these patients. Moreover, the implications of this study are beyond HD patients and can be implicated in all clinical states associated with primed PMNLs and accelerated atherosclerosis such as hypertension and diabetes.


Telocytes (TCs) are a distinct type of interstitial cells characterized by a small cell body and extremely long and thin telopodes (Tps). The presence of TCs has been documented in many tissues and organs (go to http://www.telocytes.com). Functionally, TCs form a three-dimensional (3D) interstitial network by homocellular and heterocellular communication and are involved in the maintenance of tissue homeostasis. As important interstitial cells to guide or nurse putative stem and progenitor cells in stem cell niches in a spectrum of tissues and organs, TCs contribute to tissue repair and regeneration. This review focuses on the latest progresses regarding TCs in the repair and regeneration of different tissues and organs, including heart, lung, skeletal muscle, skin, meninges and choroid plexus, eye, liver, uterus and urinary system. By targeting TCs alone or in tandem with stem cells, we might promote regeneration and prevent the evolution to irreversible tissue damage. Exploring pharmacological or non-pharmacological methods to enhance the growth of TCs would be a novel therapeutic strategy besides exogenous transplantation for many diseased disorders. History of telocyte identificationElectron microscopic features of TCsTC immunohistochemistryDistribution of TCsCellular junctions of TCs and their functional implicationsTCs in tissue repair/regenerationHeartLungs and tracheaSkeletal muscleSkinMeninges and choroid plexusEyeLiverUterusUrinary systemTargeting TCs as a potential therapeutic strategySummaryTelocytes (TCs), a novel type of interstitial cells, was firstly described in 2010 by Popescu’s group as a case of serendipity Cells with telopodes (Tps) is the shortest definition of TCs e.g. endothelial cells, which express CD34 Although TCs are positive to certain tested markers such as c-kit/CD117, vimentin, CD34 and VEGF, the positivity to these markers varies among different organs and tissues Since their identification in 2010, TCs have been found existing in fish, reptiles, mammals and humans, and have been identified in the interstitial compartment of many tissues and organs, including heart  via heterocellular junctions via paracrine and/or juxtacrine secretion of small molecules, or via shed extracellular vesicles which send important macromolecules  to neighbouring cells in normal and diseased states Multiple electron microscopy techniques, including transmission electron microscopy, electron tomography, SEM, FIB-SEM, have demonstrated that TCs have their ‘strategic’ locations in tissues Increasing evidence indicated that TCs might be implicated in tissue homeostasis and development Although the tempting idea of using stem cells and progenitor cells with regenerative potential has been hailed for many years as a promising cell-based therapeutic strategy for tissue repair/regeneration, its actual therapeutic activity is far from satisfactory versus newborns, which might be responsible for reduced cardiac regenerative capacity with ageing process Cardiac stem cells (CSC) and cardiomyocyte progenitors (CMP) are appropriate sources for regenerative medicine strategies for cellular cardiomyogenesis and neovascularization Cardiac TCs in primary culture is a useful tool to investigate cell functions, including their roles in cardiac regeneration via paracrine secretion as shown by their positivity to VEGF and NOS2 immunohistochemistry staining in vivo and the use of cardiac tissue engineering in vitro will help further clarify the functional roles of TCs in cardiac repair and regeneration Given that the potential of cardiac regeneration/repair significantly affects the consequences of myocardial infarction (MI), stem cell therapy was recognized as a prompting strategy for ischaemic cardiomyopathy Telocytes contribute to form a 3D interstitial network in pleura, trachea and lungs, through close associations with fibroblasts, smooth muscle cells, endothelial cells, immunoreactive cells and nerve endings, which suggests the conventional roles of TCs in mechanical support, immune surveillance and intercellular communication and signalling Based on the important fact that TCs connect with multiple cells in the lungs and coordinate the intercellular communications, the potential significance of TCs in the pathogenesis of certain lung diseases has been has been suggested, including pulmonary infectious diseases, chronic obstructive pulmonary disease and interstitial lung disease Adult skeletal muscle has a remarkable regenerative capacity after injury. The activation of the muscle stem cells, also called satellite cells, is a limiting step in muscle regeneration, which enables satellite cells to proliferate, migrate and differentiate into new skeletal myocytes in vitro, which might be critical for scaffold guidance of stem/progenitor cells in muscle regeneration in situ and in vitro, TCs could also be involved in angiogenesis and vascular stability during tissue repair in skeletal muscle In addition to muscle satellite cells, non-satellite cells as well as many other types of cells such as bone marrow-derived cells and pericytes contribute consistently to the extraordinary regenerative ability of skeletal muscle n = 30 for one TC, 438.6 ± 149.1 nm in diameter) have recently been identified in close vicinity of human dermal TCs The extraordinary regenerative capacity of skin is particularly important for skin repair and regeneration following injury and disease. The presence of TCs in skin has previously been documented. By transmission electronic microcopy, TCs were identified scarce in papillary dermis but numerous in reticular dermis Systemic sclerosis (SSc), a heterogenous chronic connective tissue disease, is characterized by endothelial dysfunction, immune system disorders, and progressive fibrosis in skin and visceral organs including heart, lung, kidney and oesophagus Adult neurogenesis is the process of generating new functional neurons from neural stem cells (NSC) and neural progenitor cells (NPC) to react and adapt to extra stimuli in various physiological, pathological and pharmacological conditions Stem cell therapies are considered as innovative approaches for preserving or restoring vision for patients with eye diseases Previously, the presence of TCs and stem cells in the limbus and uvea of mouse eye has been demonstrated Liver possesses an extraordinary capacity to regenerate after toxic or drug-induced injury, virus infection, ischaemia and surgical resection The potential roles of TCs in liver regeneration have recently been demonstrated by using a murine model of partial hepatectomy (PH) in vitroe.g. preeclampsia) The presence and functions of TCs in human non-pregnant and pregnant myometrium have previously been demonstrated In addition, TCs have also been identified in human endometrium tissues, indicating that TCs may support the structure of the stratum functionalis of endometrium The formation, elongation, deviation and ramification of Tps may importantly modulate the morphology and the function of TCs, as well as influence their contacts with per se or other cells. Optical stimulation and guidance of neuronal cells which aimed to modify the neuronal growth by using low-level laser stimulation (LLLS) has extensively been studied i) thinner and longer cytoplasmic prolongations; (ii) presence of dense core granules and microtubules and (iii) no peripheral actin filaments It has previously been reported that TCs are present in the upper lamina propria (ULP) of the human renal pelvis, ureter and urethra, as well as in kidney and urinary bladder via caudal vein was effective to reduce histological renal damage and attenuate renal dysfunction after renal ischaemia-reperfusion injury (IRI) in rats, which might be partially related to the secretion of growth factors other than anti-inflammatory mechanisms in vitro probably because of their insufficient paracrine ability of growth factors in such circumstance The presence of TCs in the urinary system also suggest their potential involvement in the repair and regeneration of injured tissues during diseases such as acute renal failure. Previously, it has been reported that injection of TCs via increasing cardiac angiogenesis, improving reconstruction of the TC network and decreasing cardiac fibrosis in vitroTelocytes have been found to be decreased in experimental MI, especially in fibrotic areas via transplantation of TCs and the loss-of-function assay via specific inhibition of the biological functions of TCs in animal models in vivo, as well as the primary culture of TCs from different tissues, the co-culture of TCs with other cells and the use of 3D culture environment and tissue engineering in vitro, to further confirm and clarify the mechanisms by which TCs contribute to tissue repair and regeneration. Also, a deeper understanding of the relationship between TCs and stem/progenitor cells is of great need. All these studies will be useful to clarify the biological functions of TCs in tissue repair and regeneration, as well as to provide new insights into the potential therapeutic values of TCs in regenerative medicine.In summary, we have systemically reviewed the most recent studies of the potential significance of TCs in tissue repair and regeneration in heart, lung, skeletal muscle, skin, meninges and choroid plexus, eye, liver, uterus and urinary system. However, it has to be noted that up till now, the studies of TCs were mainly based on the morphology aspect, while direct evidence to show the roles of TCs in diseases and regenerative medicine is still lacking. In such circumstances, it is necessary to explore TC-specific markers useful to identify the presence of TCs in a more specific and easier way in tissues. In addition, it is highly desirable to perform more functional studies, for example the gain-of-function assay


Congenital hearing loss affects one to three of every 1,000 live born infants ,3. To adData were gathered by using the EHDI Hearing Screening and Follow-up Survey (HSFS), which was fully implemented starting in 2006. This survey is sent annually to the EHDI program coordinator in each U.S. state, the District of Columbia, and each participating territory and freely associated state. The HSFS requests nonestimated, aggregate information about the receipt of hearing screening, diagnostic testing, and EI for every occurrent birth within the jurisdiction. The numbers of occurrent births are compared for accuracy with data from the jurisdiction’s Vital Records program and the National Vital Statistics System. Infants were classified as LFU/LTD-DX or LFU/LTD-EI if they did not receive recommended follow-up services or if they received services without the results being reported to the jurisdictional EHDI program. LTD can occur because the results of diagnostic testing and enrollment in EI are not universally required to be reported. Although strategies used to target LFU and LTD differ, these two categories are grouped together in the HSFS because it is problematic for most programs to differentiate between these different types of cases. The denominators for LFU/LTD-DX and LFU/LTD-EI used by CDC are total infants not passing the final hearing screening and total infants identified with a permanent hearing loss, respectively. More details about the HSFS and data definitions have been published ,5. The r†Data for this report are based on the HSFS conducted for the years 2006–2012, using aggregate jurisdiction-reported totals. Some jurisdictions did not respond to the HSFS in ≥1 years because completion of the survey is voluntary, the requested data were not available at the time of reporting, or another reason. Data for individual years and data at the jurisdictional level are available online.In 2012, an average of 96.6% of newborns were screened for hearing loss compared with 95.2% in 2006  and 2. OBased on available data from the HSFS, a number of jurisdictions have made progress in documenting the diagnosis of infants with permanent hearing loss and their enrollment in EI. For example, 10 jurisdictions had an improvement of at least 10% for diagnosed hearing loss among infants who did not pass the hearing screening  and 2. SImprovements in the provision and documentation of EHDI services between 2006 and 2012 have resulted in decreases in the rate of infants referred from screening and increases in infants receiving the testing needed to confirm a hearing loss. This progress has helped drive increases in the number of children reported with permanent hearing loss from 3,261 (2006) to 5,475 (2012) and an increase in prevalence from 1.1 to per 1.6 per 1,000 screened. The increase in documented cases was accompanied by a decrease in LFU/LTD-DX of 11.8% between 2006 and 2012. Similarly, the documented receipt of EI services increased by 6.3% while LFU/LTD-EI decreased by 15.7%. Other factors that contributed at least in part to this progress include 1) improvements in the functionality of state and territorial EHDI information systems, 2) increased awareness among health care providers about the importance of documenting the receipt of follow-up services, 3) continued progress by state and territorial EHDI programs in tracking infants needing follow-up services, and 4) active support by national agencies and organizations.To build on the progress already made in diagnosing and enrolling infants with hearing loss in EI services, continued work is needed to further reduce the number of infants classified as LFU/LTD each year. Unless infants with hearing loss receive recommended diagnostic and EI services, they are still at risk for avoidable delays in their speech and language development ,3. In adThis report updates an earlier summary of EHDI data during 1999–2007 that provided information on infants with hearing loss . Since t¶The findings in this report are subject to at least five limitations. First, some states and territories either did not respond to the HSFS or were only able to provide limited data in ≥1 reporting years. As a result there are differences in the number of jurisdictions reporting data each year. Second, the data reported only reflect those services that infants were documented to have received. Because reporting of newborn hearing screening and follow-up data are not required in each state and territory, it is possible for a jurisdiction to have a higher percentage of infants receiving diagnostic and EI services (and therefore lower rates of LFU/LTD) than what was reported by the HSFS. Third, there are multiple ways to calculate LFU/LTD, and the CDC definition might not fully reflect the progress jurisdictions have made in ensuring that infants receive recommended follow-up services. Fourth, there is variation between jurisdictions in the percentage diagnosed with permanent hearing loss and the reasons for this, including the impact of different screening protocols, cannot be assessed with currently available HSFS data. Fifth, all HSFS data are reported voluntarily and might include inaccuracies because some jurisdictions did not correctly report LFU/LTD and other data in accordance with the HSFS data definitions.To build on the recent improvements summarized here and ensure continued progress toward identifying and providing EI for all infants with permanent hearing loss, current practices should evolve and take advantage of new collaborations and opportunities, such as emerging technologies. Improvements in existing clinical and public health infrastructures and adoption of technologies, such as electronic health records and clinical decision support tools, can assist providers and EHDI programs in improving coordination, delivery, and documentation of recommended EHDI services –9.What is already known on this topic?Progress has been made in screening and diagnosing infants with hearing loss, reducing the number of infants lost to follow-up/lost to documentation, and increasing enrollment in early intervention. Ensuring infants receive recommended services is crucial to help prevent delays in speech, language, social, and emotional development that can occur when permanent hearing loss is not identified early.What is added by this report?Analysis of Early Hearing Detection and Intervention program survey data showed that, during 2006–2012, the number of jurisdictions reporting data increased from 49 to 57, rates of screening increased from 95.2% to 96.6%, rates of diagnosis among infants not passing the final screening increased from 4.8% to 10.3%, and enrollment in early intervention of infants diagnosed with permanent hearing loss increased from 55.4% to 61.7%, while the rates of lost to follow-up/lost to documentation declined.What are the implications for public health practice?EHDI programs should continue to work with health care providers who provide diagnostic and early intervention services to accurately document the receipt of necessary follow-up services, thereby increasing the opportunities for infants to receive proper care to minimize the negative impact that hearing loss can have on their speech, language, and emotional development.


Sphingomonas sp, strain YL-JM2C, isolated from activated sludge of a wastewater treatment plant, was very effective on degrading TCS. Response surface methodology (RSM) was applied to optimize the conditions like temperature and pH. From RSM, the optimal TCS degradation conditions were found to be 30 °C and pH 7.0. Under optimal conditions, strain YL-JM2C completely mineralized TCS (5 mg L−1) within 72 h. Gas chromatography-mass spectrometry analysis revealed that 2,4-dichlorophenol, 2-chlorohydroquinone and hydroquinone are three main by-products of TCS. Furthermore, stable isotope experimental results revealed that the 13C12-TCS was completely mineralized into CO2 and part of heavier carbon (13C) of labeled TCS was utilized by strain YL-JM2C to synthesize fatty acids (PLFAs). Cell surface hydrophobicity (CSH) and degradation test results suggested that the strain could enhance degradation capacity of TCS through increasing CSH. In addition, the bacterium also completely degraded spiked TCS (5 mg L−1) in wastewater collected from the wastewater treatment plant. Hence, these results suggest that the strain has potential to remediate TCS in the environment.Triclosan (TCS) is one of the most widespread emerging contaminants and has adverse impact on aquatic ecosystem, yet little is known about its complete biodegradation mechanism in bacteria.  Triclosan , also known as Ingrasan, is a biocide that inhibits the enoyl-acyl-carrier protein-reductase, which is an essential enzyme for the synthesis of fatty acids to develop bacterial cells4Studies suggest that TCS has weak androgenic activity against aquatic organisms and revealed both androgenic as well as estrogenic responses in human breast cancer cells61011131415161713C-labeled TCS to aid for tracking the movement of the heavier carbon (13C) from the labeled compound into the DNA (genetic biomarker) of the active TCS-utilizing microorganisms in the microbial consortia1Pseudomonas putida and Alcaligenes xylosoxidans subsp. denitrificans were reported to grow on the TCS-containing agar platesSphingomonas sp. Rd1, Nitromonas europaea, Sphingomonas sp. PH-07 and Sphingopyxis strain KCY1 were not shown to use TCS as a sole source of carbon920212124Biodegradation of triclosan in the environment and wastewater has recently attracted more attention. To date, some researches have used the stable isotope probing by 13C) from 13C12-TCS into bacterial cellular fatty acids . The changes of the bacterial CSH during biodegradation of TCS were also studied to provide a theoretical basis for the bacterial interaction of emerging contaminants like TCS in the ecosystem. In the end, the aim of this study was to address the unanswered issues for TCS biodegradation.In this study, we isolated and identified TCS-degrading bacterium strain YL-JM2C and optimized conditions for TCS degradation by Response surface methodology (RSM). We demonstrated that strain YL-JM2C could utilize TCS as the carbon source by analyzing the flow of heavier carbon , pH (X2) and biomass (X3) on TCS degradation by strain YL-JM2C. Design established conditions of variables collectively with the experimental responses are given in p < 0.05) eliminated. The following equation was obtained to clarify the TCS degradation in strain YL-JM2C.RSM based on Box Behnken design was applied to investigate the main and interactive effects of significant parameters including temperature  as well as pH (X2), the square term coefficient of temperature (X1) as well as pH (X2) and the cross-product term coefficient of temperature (X1) and pH (X2) showed significant effects (p < 0.05) on the TCS degradation by strain YL-JM2C whereas the linear term coefficient of biomass (X3) and square term coefficient of biomass (X3) were not significant (p > 0.05). As the value of inoculum biomass (non-significant variable) fixed at 0.2 (g L−1), the three dimensional response surface was plotted to directly show the effects of temperature and pH on TCS degradation in strain YL-JM2C . YL-JM2C . As showSphingomonas sp. strain YL-JM2C was grown on yeast extract (0.04%)-containing ammonium mineral salts medium (AMS) with TCS (5 mg L−1). The growth of the strain was monitored on spectrophotometer with the increase in optical density at 600 nm and the maximum absorbance of growth was recorded as 0.44. The strain degraded TCS completely within 72 h . The degradation by-products identified on the basis of retention time with respect to their mass spectrum were matched with authentic standard compounds and also reported data. The peak at retention time of 28.160 min corresponded to trimethylsilyl (TMS)-TCS and decreased over time and completely disappeared within 84 h. TCS disappeared with the release of three major intermediates , which suggested the conversion of 2-chlorohydroquinone to hydroquinone. GC-MS analysis of the sample confirmed the formation of hydroquinone.13C12-TCS, the percentage of 13C/12C of the headspace carbon dioxide (CO2) rapidly increased to about 51% within 24 h. Yet, in unlabeled TCS, the percentage of 13C/12C of the headspace CO2 remained low around 1%  after incubation with 13C12-TCS in the microcosm. Results of δ13C of PLFAs showed that the PLFAs of 14:0 2OH, 16:0, sum in feature 3, sum in feature 8 and 16:1 w5c have contained significant quantity of heavier carbon (13C) from labeled TCS  was used and no yeast extract was added throughout the experiment. By the determination of heavier carbon (13C) released from labeled TCS into CO2, we can confirm TCS is completely mineralized by strain YL-JM2C into CO2. In addition, since fatty acids are necessary for building cell membrane of living microorganisms, we have also confirmed that the 13C from labeled TCS was used by strain YL-JM2C to synthesize its cellular fatty acids. These results suggest that the bacterium is not only able to mineralize TCS into CO2 but also utilize TCS as a sole source of carbon for its biosynthesis. Due to the low solubility and antimicrobial activity of TCS, we only used 4–7 mg L−1 TCS in the medium, and this low concentration of carbon source was unable to cause significant bacterial growth observed using optical density at 600 nm (data not shown). Yeast extract was added to help strain YL-JM2C to show more significant growth in the presence of TCS. The stable isotope result demonstrated that strain YL-JM2C could mineralize and utilize TCS for biosynthesis in the absence of yeast extract. In addition, TCS was used to block an enzyme called NADH dependent enoyl-acyl carrier protein reductase (encoded by fabI gene), which blocks the synthesis of fatty acids in the bacteria. In this study, stable isotope experiment of PLFAs demonstrated that strain YL-JM2C could assimilate the carbons located in TCS into fatty acids like 14:0 2OH, 16:0, sum in feature 3, sum in feature 8 and 16:1 w5c. This result suggested that strain YL-JM2C has novel mechanism to overcome the antimicrobial activity of TCS. Future studies are needed to quantify the amount of 13C-TCS transformed into each of its by-products, calculate the concentration of 13C-TCS entering into bacterial cell biomolecules, and also identification of responsible genes for the resistance and degradation of TCS in Sphingomonas sp. strain YL-JM2C.In strain YL-JM2C, to confirm complete mineralization of TCS and also the use of TCS as a sole source of carbon for anabolism, stable isotope labeled TCS  of labeled TCS was utilized by strain YL-JM2C for fatty acids synthesis. The CSH of strain YL-JM2C increased during the degradation of TCS and the increasing CSH helped the ability of strain YL-JM2C to degrade TCS. Overall, this study suggested that strain YL-JM2C could be a good candidate for the remediation of TCS in the contaminated wastewater.In conclusion, 13C12, 99%) was purchased from Cambridge Isotope Laboratories, Inc, USA. Acetone, acetonitrile and methanol were purchased from Merck, Germany. All other chemicals were of pure analytical-grade or highest grade available commercially. Stock solution (5 g L−1) of TCS was prepared with acetone and stored in brown bottles at −20 °C before use. AMS composition: K2SO4, 0.98 mM; KH2PO4, 3.9 mM; Na2HPO4.12 H2O, 6.1 mM; (NH4)2SO4, 5.88 mM; MgSO4.7 H2O, 0.15 mM; CaSO4.2 H2O, 0.07 mM; CoMoO4, 0.004 mM; KI, 0.001 mM; ZnSO4.7 H2O, 0.002 mM; MnSO4.H2O, 0.002 mM; H3BO3, 0.002 mM; FeSO4.H2O, 0.08 mM; H2SO4, 0.1 mM. The AMS medium was set to pH 7.00 (using 2 M NaOH or 2 M H2SO4) and sterilized in autoclave at 121 °C.Irgasan  was purchased from Sigma-Aldrich Co., USA. Triclosan , through enrichment on TCS. For enrichment, 5 mL of activated sludge was added to 250 mL Erlenmeyer flask containing 95 mL of AMS with TCS (5 mg L−1) as a sole source of carbon (acetone free). The enrichment flask was incubated at 30 °C under dark condition on shaker at 150 rpm. Upon complete degradation, 5 mL of cultures were transferred to a new flask containing 95 mL of AMS with TCS (5 mg L−1). After 6 months of enrichment, one milliliter of the enrichment culture was serially diluted in R2A plates having TCS (5 mg L−1). Microorganisms grown on R2A agar plates containing TCS were further purified, and only one colony showed rapid degradation of TCS. The microorganism was designated as strain YL-JM2C. The microorganism was identified by using 16 S rRNA gene sequence analysis−1). RSM based on Box-Behnken design was used to optimize the above mentioned parameters and their interaction which significantly influenced TCS degradation in strain YL-JM2C23−1). The microorganism was inoculated into the medium and the samples were collected after 72 h to quantify the residual TCS. A three-variable Box-Behnken design consisting of 15 experimental runs with 3 replicates at the midpoint was applied in this experiment. The experimental design was provided in The experiments were conducted to elaborate important parameters like temperature, pH and biomass to get optimal condition for the degradation of TCS in strain YL-JM2C. In single-factor experiments, we studied the optimal ranges of three main variables such as temperature (20–40 °C), pH (5–9) and biomass . The bacterial growth was measured at 600 nm by UV-spectrophotometer (UV-5200 Spectrophotometer). For degradation studies, the TCS degradation was monitored by HPLC. Uninoculated culture flasks with the same concentrations of TCS were served as a control.To monitor the effects of TCS on the growth of strain YL-JM2C, strain YL-JM2C was grown in 500 mL Erlenmeyer flask containing 100 mL of yeast extract (0.04%)-containing AMS with appropriate concentration of TCS (4–7 mg L−1) were extracted and identified by GC-MS. The samples were taken at regular intervals . The same bacterial culture supernatant lacking TCS was used as a negative control, and non inoculated control containing TCS (5 mg L−1) was included as well. The collected samples were centrifuged (7000 × g) for 20 min, adjusted to pH 2.0 with 2 M H2SO4 and were extracted with ethyl acetate. For extraction, supernatant was mixed with equal volume of ethyl acetate in a brown bottle and shaken for 6 h at 150 rpm on shaker. Further, the sample was transferred to separating funnel and allowed for 15 min to separate. The ethyl acetate layer was collected and the aqueous layer was used for one more time extraction with ethyl acetate. Both ethyl acetate layers were combined together and evaporated at room temperature on anhydrous sodium sulfate. The extracted samples were reconstituted in acetone (470 μL) and derivatized with BSTFA (30 μL). The derivatized samples were analyzed by the GC-MS. The metabolites identified by mass spectrometry analysis were matched with authentic standard compounds as well as reported data.The metabolic products of TCS in strain YL-JM2C cultures containing TCS (5 mg Lp-xylene (hydrocarbon) was used for CSH determination of Sphingomonas sp. strain YL-JM2C. The CSH value was calculated using equation The relative CSH of the strain YL-JM2C during the growth and biodegradation of TCS was assessed by the microbial adherence to hydrocarbon methodI and F represent the initial and the final optical densities at 600 nm of the aqueous phase.−1) were harvested by centrifugation , washed and resuspended in 50 mM phosphate buffer (pH 7.0). The cell-free extract were prepared through ultrasonication  followed by centrifugation . The supernatant obtained was used as the crude enzyme for enzyme assaysThe cells of strain YL-JM2C grown on yeast extract (0.04%)-containing AMS medium supplemented with TCS (5 mg L13C12-TCS was used to confirm TCS carbon utilization by the isolated bacterium strain YL-JM2C. Fifty milliliters of sterile ASM (without yeast extract) containing 13C12-TCS (5 mg L−1) was filled into a 120 mL serum bottle. Artificial air  was used to purge ASM to remove CO2. The serum bottle was then inoculated with strain YL-JM2C  and sealed by butyl rubber immediately. Meanwhile, unlabeled-TCS with the same concentration was also used in another set of bottles for comparison. Heat-killed culture (YL-JM2C) and uninoculated serum bottles, which were set up under identical conditions to those of the experimental cultures and served as controls. All treatments were incubated at 30 °C under dark conditions and kept on shaker at 150 rpm. 13C/12C ratio of the headspace CO2 was determined by SIRMS at 0, 7, 14, 25, and 48 h. Thereafter the bacterial cultures were harvested by centrifugation and washed twice with phosphate buffer. Bacterial fatty acids were extracted according to the manual of MIDI Sherlock Microbial Identification System and analyzed by SIRMS immediately. SIRMS conditions were described in the SI. The percentage of added 13C incorporated into a specific PLFA was calculated as: %13C incorporation = 100 × ((Flabeled − Funlabeled) × (PLFAi)labeled]/[13C added], with the concentrations of PLFA and 13C added in mg C kg−1 solution and F value as: F = 13C/(13C + 12C) = R/(R + 1). The carbon isotope ratio (R) was evaluated from δ13C values as: R = (δ13C/1000 + 1)RVPDB, with Pee Dee Belemnite standard RVPDB = 0.011213C incorporated into an individual PLFA of labeled PLFAs was expressed as: %13C incorporation = 100 × (13CiPLFA/Σ 13Ci)PLFA, where 13CiPLFA was the amount of 13C incorporated into the PLFA of “i”, which was calculated as: 13CiPLFA = (Flabeled − Funlabeled) × [PLFAi]labeled−1, respectively, whereas the TN, TP and TCOD of collected effluent wastewater were 15.6, 1.4 and 43.0 mg L−1, respectively. The pH of the samples was adjusted to pH 7.00 (using 1 M NaOH or 1 M H2SO4). For microcosm studies, the following four experiments with TCS (5 mg L−1) spiked wastewater were conducted: (I) test microcosm with sterile influent in which cells of strain YL-JM2C were inoculated (2.66 × 107 CFU per 100 mL of wastewater) in the autoclaved influent; (II) test microcosm with non sterile influent in which cells of strain YL-JM2C were inoculated in natural influent; (III) test microcosm with non sterile effluent in which cells of strain YL-JM2C were inoculated in natural effluent; and (IV) heat-killed controls consisted of autoclaved cultures and non-inoculated controls without Sphingomonas sp. strain YL-JM2C, were set up under identical conditions to those of the experimental cultures at 30 °C.Wastewater samples were collected from raw sewage (influent) and treated wastewater (effluent) of a WWTP, Xiamen (China). The total nitrogen (TN), total phosphorus (TP) and total chemical oxygen demand (TCOD) of collected influent wastewater were 33.8, 2.5 and 95.6 mg L13C12-TCS residues at different intervals were determined by HPLC . Chloride ion analysis was carried out using Ion Chromatography. Detailed description of the analytical methods is provided in SI.Metabolites of TCS in strain YL-JM2C were analyzed by GC-MS. TCS and How to cite this article: Mulla, S. I. et al. Characterization of triclosan metabolism in Sphingomonas sp. strain YL-JM2C. Sci. Rep.6, 21965; doi: 10.1038/srep21965 (2016).


To estimate the extent to which exposure to music through earphones or headphones with MP3 players or at discotheques and pop/rock concerts exceeded current occupational safety standards for noise exposure, to examine the extent to which temporary and permanent hearing-related symptoms were reported, and to examine whether the experience of permanent symptoms was associated with adverse perceived general and mental health, symptoms of depression, and thoughts about suicide.A total of 943 students in Dutch inner-city senior-secondary vocational schools completed questionnaires about their sociodemographics, music listening behaviors and health. Multiple logistic regression analyses were used to examine associations.About 60% exceeded safety standards for occupational noise exposure; about one third as a result of listening to MP3 players. About 10% of the participants experienced permanent hearing-related symptoms. Temporary hearing symptoms that occurred after using an MP3 player or going to a discotheque or pop/rock concert were associated with exposure to high-volume music. However, compared to participants not experiencing permanent hearing-related symptoms, those experiencing permanent symptoms were less often exposed to high volume music. Furthermore, they reported at least two times more often symptoms of depression, thoughts about suicide and adverse self-assessed general and mental health.before permanent and irreversible hearing-related symptoms occur, preventive measurements concerning hearing health are needed.Risky music-listening behaviors continue up to at least the age of 25 years. Permanent hearing-related symptoms are associated with people’s health and wellbeing. Participants experiencing such symptoms appeared to have changed their behavior to be less risky. In order to induce behavior change  According to the World Health Organization, adult onset hearing loss (HL) is the second leading cause of ‘years lived with disability’ (YLD) after depression at global level, accounting for 4.6% of total global YLDs Outside the workplace millions of adolescents and young adults are potentially at risk of permanent hearing symptoms through listening to their favorite music. The risks of excessive music-listening are most likely to result in hearing-related symptoms such as tinnitus – a perceived sound in the ears with no external source of sound being present It has been estimated that about 20% of Dutch adolescents aged 12–16 years are potentially at risk of developing hearing-related symptoms after 5 years because of listening to potentially hazardous music levels. Between 30.0% and 61.2% of them reported temporary hearing-related symptoms such as tinnitus after exposure to music from MP3 players and at discotheques Symptoms of poor hearing may lead to difficulties in future life. Because HL may influence communication and behavioral skills, it can adversely affect education and quality of life. Also, it is a growing social problem as more and more young people are limited in their choice of or even rejected from jobs because of preventable HL. Furthermore, it may lead to reduced psychological and social function, such as increased feelings of isolation, depression, loneliness, anger, fear, frustration, stress and disappointment Among 12–16 year olds, mainly adolescents attending pre-vocational education report relatively high levels of music exposure According to the Dutch Act on public health, the Municipal Executive has to acquire, based on epidemiological analysis, insight into the health situation of the population and has to take care of systematic monitoring and highlighting of developments in the health status of young people A total of 1228 students (aged about 16–25 years) of 2 Dutch inner-city senior-secondary vocational schools were invited to complete questionnaires  about thSociodemographic characteristics included sex, age, ethnicity  and home situation (living with parent(s); not living with parent(s)). Ethnicity was determined on the basis of mother’s and father’s country/countries of birth according to definitions of Statistics Netherlands Estimation of risky music listening behaviors – MP3 player listening and discotheque and pop/rock concert attendance – previously has been described in detail Since we did not measure music volume levels, to be able to estimate the exposure to potentially hazardous music levels, we had to estimate the music-volume levels per music source. Portnuff and Fligor have evaluated the output levels of several most popular players Within current European occupational safety standards (week) = 56/2(L-80)/3; L stands for the estimated dBA level To be able to estimate a weekly music dose (D) on the basis of reported exposure times and levels, we first calculated permissible exposure limits (PELs) for the estimated dBA levels of each participant per music source (MP3-player use and discotheque and pop/rock concert attendance) using the equation: PELTo evaluate potential risk behavior, we first dichotomized responses on the basis of the loosened safety standard into students who were estimated to not be exposed to potentially hazardous music levels  and those who were estimated to be exposed to potentially hazardous music levels  Hearing symptoms included symptoms such as tinnitus, muffled sounds, distortion, hyperacusis or (temporary) hearing loss. Temporary hearing-related symptoms were categorized as ‘I experienced hearing-related symptoms at least once in the past month after listening to an MP3 player’  and ‘I experienced hearing-related symptoms at least once in the past year after visiting discotheques and/or pop/rock concerts’ . Permanent hearing-related symptoms were categorized as ‘I am constantly experiencing hearing symptoms’ .‘General health’ was categorized as: ‘My health in general is good or very good’ (yes/no). ‘Mental health’ was measured with the five-item Mental Health Inventory (MHI-5) ‘Symptoms of depression’ were measured with the Center for Epidemiologic Studies Depression Scale (CES-D) p values of <0.05 were considered to be statistically significant.Cross-sectional statistical analyses were performed using the SPSS program . Frequency tables were used to explore music listening, risky music listening and health of the total study population (N = 943), and females (n = 593) and males (n = 350); frequency differences were examined through chi-square statistics. Multivariate odds ratios (ORs) and their 95% confidence intervals (CIs) were calculated with multiple logistic regression analyses to explore the association between the estimated exposure to high-volume levels and both reported temporary and permanent hearing-related symptoms. Frequency tables and multiple logistic regression analyses were used to explore the associations between permanent hearing-related symptoms and the other health indicators . Any We estimated that 19.2% of participants were exposed to high-risk sound levels; they exceeded a sound level equivalent to 90 dBA for 56 hours per week for all sources of music combined; with regard to MP3 players alone, that figure was 15.4%, and for discotheques and pop/rock concerts 3.1% .After listening to music on MP3 players 14.5% experienced temporary hearing-related symptoms; after visiting discotheques and/or pop/rock concerts 44.2%. Almost 10% of participants reported constant hearing-related symptoms; about one third of them consulted a physician for their hearing problems. Regarding the other four health indicators,  frequencies for experiencing adverse outcomes ranged between 17.8% (thoughts about suicide) and 34.5% . For all indicators, in comparison with males, females reported more often adverse outcomes .Temporary hearing-related symptoms – such as tinnitus, muffled sounds, distortion, hyperacusis or hearing loss – that occurred after using an MP3 player or going to a discotheque or pop/rock concert were associated with exposure to high-volume music . StudentCompared to students experiencing such symptoms, students not experiencing permanent hearing-related symptoms listened more than 2.5 times more often to sound levels equivalent to ≥90 dBA for 56 hours per week (high-risk sound levels) .All other health indicators – adverse perceived general health, adverse mental health, depressive symptoms and thoughts about suicide – were significantly related to permanent hearing-related symptoms (corrected for sociodemographics). Compared to participants not experiencing permanent hearing-related symptoms, those experiencing such symptoms reported at least two times more often symptoms of depression, thoughts about suicide and adverse self-assessed general and mental health .An alarming percentage of almost 10% of this relatively young age group reported permanent hearing-related symptoms. Furthermore, experiencing such symptoms was related to adverse self-assessed general and mental health, and to having symptoms of depression and thoughts about suicide.Our study is one of the first to provide a preliminary insight into the association between exposure of 15–25-year-old inner-city youth attending lower education to potentially hazardous music and temporary and permanent hearing symptoms, and into the association between permanent hearing-related symptoms and adverse health consequences. The results show that risky music-listening behaviors continue up to at least the age of 25 years among students attending lower educational levels. We estimated that by listening to high-volume music during leisure time, about 20% listened on average to sound levels that may cause permanent hearing-related symptoms such as tinnitus or even permanent hearing loss that is noticeable to individual people themselves after 5 years of such exposure As found previously Remarkably, most of the alarming percentage of almost 10% of this relatively young age group that reported permanent and thus irreversible hearing-related symptoms appeared to have changed their behavior: the more often permanent hearing-related symptoms were reported, the lower the reported average sound levels. Probably these people adjusted their behavior in order to prevent the symptoms from worsening and protect their hearing from further deterioration. Furthermore, experiencing permanent symptoms was related to adverse self-assessed general and mental health, and to having symptoms of depression and thoughts about suicide. These results show that permanent hearing-related symptoms occur considerably among certain groups of youth and are associated with young people’s health and wellbeing comparable to what has been found among adults There is previous evidence that hearing problems are associated with depression and other serious mental health problems. An Australian study found that compared with population norms, hearing disability at all levels was associated with poorer mental health before permanent and irreversible hearing-related symptoms occur, we recommend that health authorities, those involved in presenting music venues, schools, parents, and young people should be informed about the potential risks of high-volume music via public health campaigns and the mass media, and should be alerted to the need for preventive hearing healthcare by health professionals such as practitioners of pediatrics, family medicine, audiology and youth health care. Real examples of peer-group people who had lost hearing through listening to loud music should be provided On the other hand, previous studies have reported a reduction in depressive symptoms of people using hearing aids This study has several limitations. One was the use of a convenience sample of students attending senior-secondary vocational education. However, most of the characteristics of the study group reflected those of this subgroup of Dutch inner-city adolescents and emerging adults. The group of non-Dutch ethnicity (66.3%) consists of the following ethnicities: Surinam  Antillean  Moroccan (12%) Turkish  other (15%). These percentages reflect those of the population of the Rotterdam inner-city senior-secondary vocational schools. Although the proportion of females attending inner-city senior-secondary vocational education is somewhat greater than the proportion of males, the proportion of females was relatively greater in our sample We did not report on the relation between drug use and both auditory symptoms and the other adverse health indicators. Drug use might increase the risk of noise induced hearing loss and/or other adverse health indicators We may have underestimated the exposure levels for several reasons. First, because we did not take into account other potentially hazardous sounds to which the participants in our study may have been exposed, which increase the risk of hearing loss. In a previous study we found that adolescents were also exposed to other potentially hazardous sounds, such as music through loudspeakers (88.0%) and through their own activities as musicians (21.5%), as well as the noise to which they were exposed when riding mopeds or scooters (27.1%) or when using firecrackers (60.8%) In conclusion, as hearing problems tend to isolate people from friends, family and others because of a decreased ability to communicate, the impact of HI may be profound as it can impose a heavy social and economic burden on individuals, families, communities, and countries Table S1Survey items.(DOC)Click here for additional data file.