The role and extent of horizontal gene transfer (HGT) in eukaryotes are hotly disputed topics that impact our understanding of the origin of metabolic processes and the role of organelles in cellular evolution. We addressed this issue by analyzing 10 novel Cyanidiales genomes and determined that 1% of their gene inventory is HGT-derived. Numerous HGT candidates share a close phylogenetic relationship with prokaryotes that live in similar habitats as the Cyanidiales and encode functions related to polyextremophily. HGT candidates differ from native genes in GC-content, number of splice sites, and gene expression. HGT candidates are more prone to loss, which may explain the absence of a eukaryotic pan-genome. Therefore, the lack of a pan-genome and cumulative effects fail to provide substantive arguments against our hypothesis of recurring HGT followed by differential loss in eukaryotes. The maintenance of 1% HGTs, even under selection for genome reduction, underlines the importance of non-endosymbiosis related foreign gene acquisition. Eukaryotes transmit their nuclear and organellar genomes from one generation to the next in a vertical manner. As such, eukaryotic evolution is primarily driven by the accumulation, divergence , fixation, and loss of gene variants over time. In contrast, horizontal  gene transfer (HGT) is the inter- and intraspecific transmission of genes from parents to their offspring. HGT in Bacteria  and ArchCoding sequences in eukaryotic genomes that share more than 70% amino acid sequence identity to prokaryotic homologs are most likely assembly or annotation artifacts.')  . Howeverfacts.') , this reCyanidioschyzon merolae 10D  as model organisms. The Cyanidiales comprise a monophyletic clade of polyextremophilic, unicellular red algae (Rhodophyta) that thrive in acidic and thermal habitats worldwide  . With a olae 10D , Galdierria 074W , and Gala DBV009 . The majtrations . In suchtrations , althougtrations . The inttrations  underlintrations . For thiIt should be appreciated that the correct identification of HGTs based on sequence similarity and phylogeny is rarely trivial and unambiguous, leaving much space for interpretation and erroneous assignments. In this context, previous findings regarding HGT in Cyanidiales were based on single genome analyses and have therefore been questioned .Many potential sources of error need to be excluded during HGT analysis, such as possible bacterial contamination in the samples, algorithmic errors during genome assembly and annotation, phylogenetic model misspecification, and unaccounted for gene paralogy . In addiHere, we used multi-genomic analysis with 13 Cyanidiales lineages  from nine geographically isolated habitats. This approach increased phylogenetic resolution within Cyanidiales to allow more accurate assessment of HGT while avoiding many of the above-mentioned sources of error. The following questions were addressed by our research: (i) did HGT have a significant impact on Cyanidiales evolution? (ii) Are previous HGT findings in the sequenced Cyanidiales genomes an artefact of short read assemblies, limited genome databases, and uncertainties associated with single gene trees, or do they hold up with more extensive sampling? (iii) And, assuming that evidence of eukaryotic HGT is found across multiple Cyanidiales species, are cumulative effects observable, or is DL the better explanation for these results?C. merolae Soos, the species with the most limited coding capacity (4406 protein sequences), includes 89.5% of the eukaryotic BUSCO dataset. The number of contigs obtained from the Galdieria genomes ranged between 101–135. G. sulphuraria 17.91 (a strain different from the ones sequenced) was reported to have 40 chromosomes, and strains isolated from Rio Tinto (Spain), 47 or 57 chromosomes  of the C. merolae 10D telomere-to-telomere assembly. Whole genome alignments indicate that a portion of the assembled contigs represent complete chromosomes.Genome sizes of the 10 targeted Cyanidiales  range fromosomes . Pulsed- in size . The genPorphyra umbilicalis  and Galdieria lineages with 44–54 HGT events . In comparison to previous studies .The most commonly used approach to identify HGT candidates is to determine the position of eukaryotic and non-eukaryotic sequences in a maximum likelihood tree. Using this approach, 96 OGs were identified in which Cyanidiales genes shared a monophyletic descent with prokaryotes, representing 1.06% of all OGs. A total of 641 individual Cyanidiales sequences are considered as HGT candidates . The amo studies , no evid studies  did not rbcL and 18S rRNA gene markers prior to sequencing. No instances of contamination were found. Furthermore, our work relied on PacBio RSII long-read sequencing technology, whereby single reads frequently exceed 10 kbp of DNA. Given these robust data, we also tested for co-occurrence of HGT gene candidates and ‘native’ genes in the same read. The protein sequences of each species were queried with tblastn  against a database consisting of the uncorrected PacBio RSII long reads. This analysis showed that 629/641 (98.12%) of the HGT candidates co-localize with native red algal genes on the same read . It should be noted that the 10 novel genomes we determined share HGT candidates with C. merolae 10D, G. sulphuraria 074W, and G. phlegrea DBV009, which were sequenced in different laboratories, at different points in time, using different technologies, and assembly pipelines. Hence, we consider it highly unlikely that these HGT candidates result from bacterial contamination. As the accuracy of long read sequencing technologies further increases, we believe this criterion for excluding bacterial contamination provides an additional piece of evidence that should be added to the guidelines for HGT discovery . HGT-derived genes initially retain characteristics of the genome of the donor lineage. Consequently, the passage of time is required (and expected) to erase these differences. Therefore, we searched for differences in genomic features between HGT candidates and native Cyanidiales genes with regard to: (1) GC-content, (2) the number of spliceosomal introns and the exon/gene ratio, (3) differential transcription, (4) percent protein identity between HGT genes and their non-eukaryotic donors, and (5) cumulative effects as indicators of their non-eukaryotic origin .Galdieria species showed significant differences  in percent GC-content between native sequences and HGT candidates  in comparison to the majority of thermophilic organisms that exhibit higher values (~55%). On average, HGT candidates in Galdieria display 1% higher GC-content in comparison to their native counterparts. No significant differences were found for C. merolae 10D and C. merolae Soos in this respect. Because native Cyanidioschyzon genes have an elevated GC-content (54%–56%), this makes it difficult to distinguish between them and HGT-derived genes (Appendix 3).All 11 ndidates . SequencGaldieria lineage. On average, 42% of the Galdieria HGT candidates are single-exon genes, whereas only 19.2% of the native gene set are comprised of single-exons. This difference is significant  in all Galdieria species except G. sulphuraria SAG21.92  and none of its HGT candidates has gained an intron. Enrichment testing is not possible with these small sample sizes (Appendix 4).Bacterial genes lack spliceosomal introns and therefore the spliceosomal machinery. Consequently, genes acquired through HGT are initially single-exons and may acquire introns over time due to the invasion of existing intervening sequences. We detected significant discrepancies in the ratio of single-exon to multi-exon genes between HGT candidates and native genes in the SAG21.92 . The Cyaachinery , therefoGaldieria lineage . HGT candidates in Galdieria have 0.97–1.36 fewer exons per gene in comparison to their native counterparts. Because the multi-exonic HGT subset in both Cyanidioschyzon species combined includes only one multi-exonic HGT candidate, no further analysis was performed (Appendix 4).We analyzed the number of exons that are present in multi-exonic genes and obtained similar results for the  lineage . All GalG. sulphuraria 074W .Several RNA-Seq datasets are publicly available for ria 074W  and C. molae 10D . We aligolae 10D  the tranolae 10D  for botholae 10D . The aveGaldieria lineage (62.5%), 23 are exclusive to the Cyanidioschyzon lineage (24%), and 13 are shared by both lineages (13.5%)   and 45.1%  for those OGs exclusive to the Galdieria lineage. According to the HGT view, these subsets of candidates were horizontally acquired either in the Cyanidioschyzon lineage, or in the Galdieria lineage after the split between Galdieria and Cyanidioschyzon. DL would impose gene loss on all other eukaryotic lineages except Galdieria or Cyanidioschyzon. Over time, sequence similarity between the HGT candidate and the non-eukaryotic donor is expected to decrease at a rate that reflects the level of functional constraint. The average PID of ‘ancient’ HGT candidates shared by both lineages . Therefore, neither Cyanidioschyzon nor Galdieria specific HGTs, or HGTs shared by all Cyanidiales, are significantly more, or less, similar to their potential prokaryotic donors. We also addressed the differences in PID within the three groups. The average PID within HGT gene candidates of the 13 OGs shared by all Cyanidiales is 75.0%   and 75.0%  for those OGs exclusive to the Galdieria lineage. Because we sampled only two Cyanidioschyzon species in comparison to 11 Galdieria lineages that are also much more closely related  . Consequ= 65.4%) . From thears ago ) is ~5% = 90.9%) . Similar related , a compa related . Hence, Galdieria and Cyanidioschyzon, we found two cases , the second was retained by Cyanidioschyzon (and shares sequence similarity with a different group of prokaryotes), and a third paralog was retained by all other eukaryotes. It should be noted that the ‘other eukaryotes’ do not always cluster in one uniformly eukaryotic clade which increases the number of required paralogs in LECA to explain the current pattern. Furthermore, some paralogs could also have already been completely eroded and do not exist in extant eukaryotes. Similarly, 6/60 Galdieria specific OGs also contain Cyanidioschyzon genes . Here, they are nested within other eukaryote lineages and would not be derived from HGT  which cluster within the eukaryotic branch and are not monophyletic with Cyanidioschyzon HGT candidates . Approximately 1/3 of the native OGs (36.1%) has been affected by gene erosion to such a degree that it is present in only one, or two Galdieria strains. In comparison, 26.7% of the candidate HGT-impacted OGs are encoded in >10 Galdieria strains, whereas 53.0% are present in less than three. The latter number might be an underestimation due to the strict threshold for HGT discovery which led to the removal of HGT candidates that were singletons. The HGT distribution is therefore skewed towards OGs containing only a few or one Galdieria species as the result of recent HGT events that occurred; for example after the split of G. sulphuraria and G. phlegrea. In spite of the strong erosion which would also lead to partial fixation of presumably recent HGT events, we analyzed whether the distribution patterns of HGT candidates across the sequenced genomes reflect the branching pattern of the species trees , or differentially lost in one of the two lineages (according to DL). In the 60 Galdieria specific OGs we found 12 OGs containing less than 10 and more than one Galdieria species . In the remaining OGs, the HGT gene candidate is distributed across the Galdieria lineage and conflicts with the branching pattern of the species tree. HGT would assume either multiple independent acquisitions of the same HGT candidate, or partial fixation of the HGT candidate in the lineage, while still allowing for gene erosion. According to DL, these are the last existing paralogs of an ancient gene, whose erosion within the eukaryotic kingdom is nearly complete. However, it must be considered that in some cases, DL must have occurred independently across multiple species in a brief of time after the gene was maintained for hundreds of millions of years across the lineage . This implies that the gene was present in the ancestor of the Galdieria lineage and also in the last common ancestor of closely related G. sulphuraria MS1, G. sulphuraria 074W and G. sulphuraria RT22 (as well as G. sulphuraria SAG21) and the last common ancestor of closely related G. sulphuraria MtSh, G. sulphuraria Azora and G. sulphuraria YNP5587.1 (as well as G. sulphuraria 5572). A gene that was encoded and maintained since LECA, was lost independently in 6/8 species within the past few million years.As already noted above, only 50/96 of the sampled HGT-impacted OGs do not appear to be affected by erosion. Dense sampling of 11 taxa within the  dataset . Only 52es trees . This is species . In 5/12Galdieria species), OG0002676 , OG0006191 , OG0008680 , and OG0008822 . Moreover, we find 73 OGs with eukaryotes as sisters sharing over 70% PID to non-eukaryotic sequences (0.8% of the native OGs)  . On closGaldieria species, but is ancient due to its presence in G. sulphuraria and G. phlegrea). In the case of DL, no cumulative effects as well as no differences between the HGT and native dataset are expected because the PID between eukaryotes and non-eukaryotes is irrelevant to this issue because all genes are native and occurred in the LECA. According to DL, the monophyletic position of Cyanidiales HGT candidates with non-eukaryotes is determined by the absence of other eukaryotic orthologs (given the limited current data) and may be the product of deep branching effects.We assessed our dataset for evidence of cumulative effects within the candidate HGT-derived OGs. If cumulative effects were present, then recent HGT candidates would share higher similarity to their non-eukaryotic ancestors than genes resulting from more ancient HGT. Hence, the fewer species that are present in an OG, the higher likelihood of a recent HGT . This was not the case in the ‘HGT’ subset. Here, no general trend was observed .Second, we assessed if OGs containing fewer Galdieria species). This analysis revealed a significantly higher PID with non-eukaryotic sequences in favor of the HGT subset in OGs containing either one Galdieria sequence, or all 11 Galdieria sequences   . Hence, Sulfobacillus thermosulfidooxidans (Firmicutes), a mixotrophic, acidophilic (pH 2.0), and moderately thermophilic (45°C) bacterium that was isolated from acid mining environments in northern Chile  was most prominent amongst the prokaryotic orthogroups. Sulfobacillus thermosulfidooxidans shares monophyly in 6/96 HGT-derived OGs and is followed in frequency by several species that are either thermophiles, acidophiles, or halophiles and share habitats in common with Cyanidiales . The most frequent terms were: ‘decanoate-CoA ligase activity’ , ‘oxidation-reduction process’ , ‘transferase activity’ , ‘carbohydrate metabolic process’ , ‘oxidoreductase activity’ , ‘methylation ‘, ‘methyltransferase activity’ , ‘transmembrane transporter activity’ , and ‘hydrolase activity’ . In comparison to previous studies, our analysis did not report a significant enrichment of membrane proteins in the HGT dataset  . As suchG. sulphuraria have shown an increased efficiency and speed regarding the biotransformation of HgCl2 compared to eukaryotic algae ) is imported into the cell by high-affinity Pi transport systems  of a bacterial arsC gene, thus enabling the reduction of As(V) to As(III) using thioredoxin as the electron acceptor. It is known that As(III) can be converted into volatile dimethylarsine and trimethylarsine through a series of reactions, exported, or transported to the vacuole in conjugation with glutathione. Two separate acquisitions of a transporter annotated as ArsB are present in G. sulphuraria RT22 and G. sulphuraria 5572 , as well as a putative cytoplasmic heavy metal binding protein (OG0006191) in the Cyanidioschyzon lineage.Geothermal environments often contain high arsenic (Ar) concentrations, up to a several g/L as well as high levels of mercury (Hg), such as >200 µg/g in soils of the Norris Geyser Basin  and volcanic waters in southern Italy , both knic algae . Ortholo systems , whereas systems . Galdiert efflux . ArsenicGaldieria specific 4-nitrophenylphosphatases likely involved in the bioremediation of highly toxic hexachlorocyclohexane (HCH)  involved in styrene degradation and three  ne (HCH) , or morexygenase  oxidizin thought .•O2-), enzymes such as superoxide dismutase enhance the conversion of 2 x •O2-, into hydrogen peroxide (H2O2) which is in turn reduced to H2O through the glutathione-ascorbate cycle. Other toxic hydroperoxides (R-OOH) can be decomposed various peroxidases to H2O and alcohols (R-OH) at the cost of oxidizing the enzyme, which is later recycled (re-reduced) through oxidation of thioredoxin  in the Galdieria lineage  , such as reactive oxygen species (ROS) and reactive nitrogen species (RNS), for example during cellular respiration . Furtheroredoxin . The glu0004392) . In addithaliana .G. sulphuraria is able to grow heterotrophically using a large variety of different carbon sources and compounds released from dying cells (C. merolae is strictly photoautotrophic (G. sulphuraria can be maintained on glycerol as the sole carbon source (G. sulphuraria RT22 (OG0006482), G. sulphuraria Azora and G. sulphuraria SAG21 (OG0005235). The putative HGT glycerol transporters found in G. sulphuraria 074W did not meet the required threshold of two Cyanidiales sequences (from different strains) in one OG. In addition, another MIP family aquaporin, permeable to H2O, glycerol and other small uncharged molecules (G. sulphuraria Azora (OG0007123). This could be an indication of a very diverse horizontal acquisition pattern regarding transporters. OG0003954 is the only exception to this rule, because it is present in all Galdieria lineages and is orthologous to AcpA|SatP acetate permeases involved with the uptake of acetate and succinate . The OG0005153 encodes a glycosyl transferase family one protein involved in carbon metabolism . All Galdieria have an alpha-xylosidase resembling an extremely thermo-active and thermostable α-galactosidase (OG0001542) (We found evidence of saprophytic adaptations in e enzyme . This ene enzyme  as well e enzyme  which is archaea  (G. sulp0001542) . The onl3-⇌ ADP + Orthophosphate + Malonyl-CoA]. Whereas OG0002051 is present in all Cyanidiales and located in the cytoplasm, OG0007550 and OG0007551 are unique to C. merolae Soos and annotated as ‘chloroplastic'. Prior to fatty acid (FA) beta-oxidation, FAs need to be transformed to a FA-CoA before entering cellular metabolism as an exogenous or endogenous carbon source (eicosanoid metabolism is the exception). This process is initiated by long-chain-fatty-acid-CoA ligases/acyl-CoA synthetases (ACSL)  is the rate limiting and step in fatty acid biosynthesis. The bacterial ACCase complex consists of three separate subunits, whereas the eukaryotic ACCase is composed of a single multifunctional protein. Plants contain both ACCase isozymes. The eukaryotic enzyme is located in the cytosol and a bacterial-type enzyme consisting of four subunits is plastid localized. Three of the HGT orthogroups  were annotated as bacterial biotin carboxyl carrier proteins (AbbB/BCCP), which carry biotin and carboxybiotin during the critical and highly regulated carboxylation of acetyl-CoA to form malonyl-CoA  5) In the absence of detectable cumulative effects – which should definitely exist given that HGT appears to be real – the dynamics of gain and loss should be studied in more detail, first of all by producing heatmaps analogous to figure 2 but instead showing (a) sharing of putative HGT OGs and (b) sequence identity amongst putatively shared HGT OGs. One hypothesis for both why few close matches are found is that the sequences undergo accelerated evolution upon arrival and therefore it should be interesting to compare the rate of sequence evolution between the core genome and these elements.We thank the reviewer for this suggestion. To address this point, we have generated new figure, Appendix 5—figure 1. Here we show the% Protein Identity between best prokaryotic Blast-hit with each of the Cyanidiales species analyzed in our study. We appreciate the complex nature of sequence evolution after HGT, leading potentially to an accelerated divergence rate due to adaptation to a new genome and to any proteins that interact with the transferred gene product, as is found for symbiont encoded or transferred genes in any long-term interaction that can confound easy interpretation. Therefore, we present these data but did not look at this aspect in detail. We also note that the level of PID is high enough (35-45% in Appendix 5—figure 1) that we will pick up almost all of the candidates using standard BLAST. It is also hard to prove a negative result; i.e., we did not find the HGT because it is too diverged. Building HMMs for each HGT to scan all genomes may address this issue but would, in our opinion constitute a significant effort that focuses on post-HGT gene divergence and goes beyond the goals of this manuscript.6) It would be interesting to extend the analysis separately to singletons, given that many of them can be presumably verified as real based on all the other criteria that the authors use and this should allow identification of recently acquired genes, some of which may show higher homology to prokaryotic homologues. If a set of high-quality singletons could be developed, this would allow much more to be said about the overall dynamic principles involved in gene gain and loss.We respectfully disagree with this point. We intentionally avoided the inclusion of singletons because trees with singletons are never as stable as when there are multiple taxa that contain an HGT candidate. We do however take the reviewers point that a set of singletons might be useful resource for future work by others. We hence have generated a set of candidate singletons (see Appendix 5—figure 1) and made it available for download from <http://porphyra.rutgers.edu/bindex.php>.7) Sequence alignment and tree files must be included for all species and gene tree analyses within the manuscript, and made publicly available as an online resource, in addition to those genome and annotation files already identified as being deposited.Agreed. We have now made all of the requested data available for download from: http://porphyra.rutgers.edu/bindex.php.The following points should be given considerationAccelerated evolution could be studied in more detail by looking at patterns of homology within genes, to see if particular trends could be found, e.g. in terms of particular biases that reflect adaptation to eukaryotic context.We agree that these would be interesting aspects to study. We do however feel that these aspects are beyond the scope of the current study (see comments above).Differential transcription. You show statistically significant differential expression of HGT genes under temperature changes. However, the test of differential expression they perform for all analyses compares HGT genes en masse to the overall expression of the full transcriptome . If we understand this method correctly, there is a slight flaw, in that HGT genes tend to be operational genes, which are more likely to be differentially expressed under different conditions. However, many genes in the transcriptome are housekeeping genes that would not be expected to be as responsive to differential expression. In order to account for this bias, differential expression of HGT genes should be compared to other genes only across major functional categories, to show that HGT has higher or lower expression than expected with respect to expected differential expression observed across gene functional categories. This will more clearly isolate the HGT-dependent signal in the data.We are not convinced that we can “correct” for operational versus housekeeping genes to address the potential bias in the HGT expression data. By using all of the data we think that we best address the ideas that HGT genes are first, expressed, and second, respond to stress. Whether they are more or less responsive than “ancestral” genes seems to us to be a less informative question. Given that the HGT genes are resident in, and have been integrated in host cell metabolism, we expect that they will behave like any other differentially expressed, stress-related gene, and therefore the meta-analysis is probably the best approach. To restate this point in another way, assuming that novel genes need first to be “domesticated” and incorporated into host biology, the observation that many HGT-derived genes are differentially expressed under stress suggests to us that they are in fact true markers of stress.To address the reviewers’ point, we have shortened the relevant section in the manuscript text.


Tens of millions of U.S. residents have a range of adverse health outcomes caused by noise exposure  would produce minimal hearing loss in 96% of the population.In an assessment of noise exposure at college basketball games, attendees wearing dosimeters at a midsized arena were exposed to average sound levels over 98 dB, with peak levels ranging from 127.5 to 138.3 dB .The 2018 SpringStyles survey included the following question related to the use of an HPD during recreational exposure to loud sounds: “In the past 12 months, how often did you wear hearing protection devices  when attending a loud athletic or entertainment event?Independent variables included sex, age, race/ethnicity, education, household income, metropolitan statistical area of residence status, presence of hearing impairment in a household member, and frequent sporting event attendance. A total of 6,357 adults answered the question concerning HPD use during a loud athletic or entertainment event. Researchers combined participant answers into three categories: never or seldom, some or about half the time, and most of the time or always; they then applied adjusted multinomial logistic regression to examine how the likelihood of wearing an HPD varied by sociodemographic factors.Overall, 81.8% of U.S. adults aged ≥18 years reported never or seldom wearing an HPD when attending a loud athletic or entertainment event . The majCompared with adults who had a bachelor’s degree or other higher education, those with a high school education or less (odds ratio [OR] = 1.7) and those with some college education (OR = 1.6) were significantly more likely to not wear HPDs . Adults In this analysis, approximately 8% of participants reported consistent use of an HPD at loud athletic or entertainment events. Approximately two thirds of adults who were more likely to wear an HPD had at least some college education, and approximately half had higher income levels. Women and older adults were significantly less likely to wear an HPD.Persons with auditory damage caused by excessive loud sound exposure often do not recognize it. An analysis of 2011–2012 data from the National Health and Nutrition Examination Survey found that one in four U.S. adults who reported excellent or good hearing had damage to their hearing suggestive of excessive exposure to loud sounds  at loud athletic or entertainment events; women and older adults were less likely to use an HPD, whereas adults with hearing impairment, or who had a hearing-impaired household member, or some college education were significantly more likely to use an HPD.Increasing awareness about the adverse health effects of excessive noise exposure and the simple preventive measures to reduce risk are needed. Health care providers can help their patients prevent or reduce the risks for noise-induced hearing loss.


To investigate the changes of thickness in each layer, the morphology and density of inner neurons in rhesus monkeys' retina at various growth stages, thus contribute useful data for further biological studies.  The thickness of nerve fiber layer (NFL), the whole retina, inner plexiform layer (IPL), and outer plexiform layer (OPL) of rhesus monkeys at different ages were observed with hematoxylin and eosin (H&E) staining. The morphology and the density of inner neurons of rhesus monkey retina were detected by immunofluorescence.  The retina showed the well-known ten layers, the thickness of each retinal layer in rhesus monkeys at various ages increased rapidly after infant, and the retina was the thickest in adulthood, but the retinal thickness stop growing in senescent. Quantitative analysis showed that the maximum density of inner neurons was reached in adolescent, and then, the density of inner neurons decreased in adults and senescent retinas. And some changes in the morphology of rod bipolar cells have occurred in senescent.  The structure of retina in rhesus monkeys is relatively immature at infant, and the inner retina of rhesus monkeys is mature in adolescent, while the thickness of each retinal layer was the most developed in the adult group. There was no significant change in senescence for the thickness of each retinal layer, but the number of the neurons in our study has a decreasing trend and the morphological structure has changed. The structural and functional complexity of retina makes it vulnerable to alterations throughout the animal life. The morphology and function of retina may change along with the age. Optical coherence tomography (OCT) has indicated that nerve fibers, ganglion cells, and the inner plexiform layers are thinner in diabetic retinopathy (DR) patients –3. RetinThe retina of rodent models has provided an invaluable tool to study the morphology and function for more than 30 years. Among these animal models, they are mainly focused upon secondary changes affecting inner retinal cells at various times  or morphIn this study, rhesus monkeys were selected as objects to investigate the structure of rhesus monkey retinas of various ages and provide a detail analysis of the changes occurring during development. This study aims at (1) investigating the changes of each layer in the retina of rhesus monkeys at various ages and (2) observing the morphology and survival of inner retinal cells at various ages, e.g., RGCs and second-order neurons in the retina of rhesus monkeys.n = 5 monkeys per group) according to age [ad libitum. All subjects underwent standard ophthalmologic examinations including refraction, orthoptic examination, IOP measurement, and slit-lamp examination. Participants had no known neurological disease, diabetes, or active cardiovascular disease. Animal welfare was conducted under the regulation of the National Institutes of Health Guide for the Care and Use of Laboratory Animals, issued by the Ministry of Science and Technology of China [In this study, 20 healthy rhesus monkeys used for this study were maintained by the National NHPs Research Center of PriMed Shines Biotech Co., which is located in Ya'an, Sichuan, Southwest China. These monkeys were divided into four groups  through the optic nerve head were processed using a microtome (2035 Biocut Leica).All animals were deeply anesthetized with 15 mg/kg of ketamine and sacrificed, the eyes were quickly enucleated and dissected, and the eye cups were placed in formaldehyde-acetic acid-alcohol (FAA) for 24 hours. The right eyes for H&E and the left eyes for immunofluorescence were paraffin embedded, and serial sections , or anti-parvalbumin antibody  to immunolabel RGCs, rod bipolar, or amacrine, respectively. Then, the sections were incubated with secondary Alexa Fluor 488 for 1.5 hours at room temperature, and DAPI was used for nuclei staining. Sections were washed, mounted, and imaged by fluorescence microscopy . PBS was treated as negative control.The samples are preincubated in 10% normal goat serum in PBS containing 1% bovine serum albumin (BSA) and 0.3% Triton X-100 for one hour at room temperature to block nonspecific binding activity. Then, the samples were incubated overnight (4°C) with anti-NeuN antibody , anti-pkc-Sections of the retina tissue stained by H&E and immunofluorescence were analyzed, and the images were captured by brightfield microscopy  to observe retinal structures. The sections were analyzed at the superior nasal side of the central retina defined as the region one-third along the length of the retina starting from the optic nerve outwards to the periphery for further morphometric analysis . MorphomP value ≤ 0.05 was considered statistically significant .Statistical analysis was presented as mean ± standard deviation (SD) using IBM SPSS Statistics . Statistical analysis was performed using one-way ANOVA. P < 0.01) thicker than that in the adolescent group. No statistical difference in the thickness of NFL was found between adults and the senescent. The above results suggested that the thickness of NFL changed obviously from adolescent to adulthood and showed that it was thickest in the adult group, but we did not find a significant change of the thickness of NFL in the senescent compared with adults. The total retinal thickness (P < 0.01) increase in the adult group, compared with the adolescent group. The mean thickness of the whole retina in the adult group had no significant difference compared with aged rhesus monkeys. The thickness of IPL  from infant to adolescent (P < 0.01) decreased when compared with the adolescent group. In addition, it is also found that the density of RGCs in the retina of senescent rhesus monkeys is significantly (P < 0.01) lower when compared with adolescent rhesus monkeys. However, no significance was found between adult and senescent rhesus monkeys.The RGCs were labeling with NeuN antibody , and theolescent . Similarα antibody, anti-parvalbumin antibody, and anti-calbindin antibody, respectively, in the INL [P < 0.01) at adolescent in the INL; then, the density of all amacrine cells decreased significantly in adult and senescent retains (α (P < 0.05) larger when compared with the infant and senescent groups.The morphological structure of rod bipolar cells , amacrin the INL  (Figure  the INL . However retains . Findingtains (α , and thetains (α  in adoleThe present study documented morphological changes in the retina at various ages through the whole life span in rhesus monkeys. Clinically, OCT is used for the quantitative detection of retinal thickness, which is widely used due to its noninvasive and contactless imaging method . In the In conclusion, certain morphological changes represented in this article have a clear physiological correlate. With age, the eye went through certain changes, including the thickness of each layer, the density, and the morphology of RGCs and second-order neurons. The maximum density of second-order neurons was reached at adolescent in the INL, while synapses and axons of inner neurons were well developed in adulthood for each layer and got to the thickest in adulthood. The above results indicate that the structure of the retina in rhesus monkeys is relatively immature at infant, and the inner retina of rhesus monkeys is mature in adolescent, while the thickness of each retinal layer was the most developed in the adult group. There was no significant change in senescent for the thickness of each retinal layer, but the number of the neurons in our study has a trend of decreasing and the morphological structure has changed. These changes may lead to more visual clarity and more complete development from infant to adolescent, but vision acuity may decline gradually from adult to senescent.


We report a case in which we quantified the maturation of the central auditory pathway in children with hearing loss and associated disabilities; the audiological intervention was performed using the BAHA softband. The hearing aid was applied according to the international clinical protocols. The presented case reveals the importance of using the P1 CAEP biomarker in clinical practice along with a neuropsychological evaluation to assess the maturation of the central auditory pathways and to objectively quantify the results of auditory rehabilitation in children with hearing loss and associated disabilities. Hearing loss, especially in children, may cause a delay in language development, and consequently, it may affect cognition and lead to learning disabilities . For thiIn the case of the auditory cortex not receiving auditory stimuli in an optimal period, then we are dealing with the phenomenon of cross reorganization  -7. 30-40The P1 CAEP biomarker is easy to record, non-invasive, and it is an objective parameter for assessing the efficacy of hearing aid use in children with hearing loss and associated disabilities .We present the case of a 38-month-old girl who was diagnosed with Goldenhar syndrome as a newborn. She was born at term following a normal pregnancy . Postnatally, she presented nasal obstruction, inspiratory dyspnea, feeding difficulties, vomiting after a feed, episodes of apnea and cyanosis after regurgitation of gastric contents. An ENT consultation was performed, and bilateral choanal atresia was identified; the surgical lysis of the imperforation was performed with stent mounting. The patient presented other malformations: microretrognathism, microcrania , mandibular hypoplasia, low ear implantation, pre-auricular appendix, external auditory canal (EAC) atresia of the left ear, short neck, bilateral microtia. Brain computed tomography performed at 23 months revealed a cleft palate in the right parasagittal plane, adenoid hyperplasia with partial choanal atresia, left EAC atresia, right EAC stenosis; also, the left ossicle chain was not identified (excepting a piece of the hammer), while the internal auditory canal (IAC) had normal morphology. At 11 months, the diagnosis of moderate conductive/mixed hearing loss in the right ear and severe mixed hearing loss in the left ear was established .Hearing rehabilitation was achieved using the BAHA Softband. The control audiogram at 38 months showed hearing thresholds at 30 dB HL in the sound field.Cortical auditory potentials were recorded at 38 months to see if the amplification made by the BAHA 5 Softband hearing aid ensured proper maturation of the central auditory pathway. Testing was carried out within 90 minutes in an anechoic room. The patient was comfortably placed in the parent’s lap and watched cartoons on mute/silent mode. The electrodes were placed on the scalp as follows: the active Cz electrode connected to the positive input of the amplifier, the reference electrode on the mastoid process, and the ground electrode at Fpz. A supraorbital electrode, paired with an infraorbital reference electrode placed ipsilaterally, was used to minimize ocular artifacts. Electrode impedance was maintained between 1-3 kOhms. A calibrated loudspeaker placed at 1 m distance in 0° angle emitted a speech stimulus, the “ba” syllable, at 70 and 80 dB nHL intensity. The stimulus rate was 1.10/s, the duration was 114875 µsec, for 512 sweeps, artifact rejection with amplitude criterion at ± 100 µV. CAEPs recorded following the delivery of the stimulus were analyzed using the SmartEP USB software provided by Intelligent Hearing System.The measurement of the P1 CAEP biomarker after 27 months of hearing aid use shows normal latency (125 ms), morphology, and amplitude of 3.31μV . This fiWe evaluated the patient using the Denver Developmental Screening Test II (DDST II) adapted for children with multiple disabilities. DDST II consists of 125 items that aim to evaluate the child in the following areas: personal-social, fine motor-adaptive, language, gross motor ability. It also includes five “behavior-during-testing” items that help the physician assess the child’s general behavior, obtaining a rough indicator of her abilities. The child’s performance is usually classified in one of three categories: “normal”, “suspect” or “untested”. The traditional scoring does not provide a quantitative estimation of development. In contrast, DDST II is specifically designed to detect varying degrees of developmental impairment, monitoring progress over time and child’s integration into specialized rehabilitation programs.Developmental testing was first performed at 32 months using the Denver Developmental Screening Test II. Subsequently, after the patient was integrated into a rehabilitation program , reevaluation was performed at 38 months .Our research is one of the few documented studies in which maturation of the central auditory pathway was evaluated in children with BAHA interventions.In our patient, the therapeutic intervention to compensate for the auditory deficit was performed early, and this allowed the central auditory pathway to develop according to her age – P1 morphology and latency proper for age . P1 can Pre- and post-intervention progress monitoring can be a difficult task for the clinician. In many cases, it is difficult to obtain objective measurements of auditory thresholds in children with multiple disabilities through audiograms with visual reinforcement because the child cannot be conditioned on the stimulus and the assessment of the perceptual language is limited (these children do not acquire open-set and close-set word recognition) .In a study performed on 146 children aged 6 to 15 with a non-syndromic cleft palate or orofacial cleft, without hearing loss, Xiaoran Ma et al. demonstrated that P1-N1 amplitude was significantly decreased, and N1 had a higher latency compared to healthy children of the same age . These rIn our patient, the developmental quotient is lower than 1, suggesting inappropriate age development. A gain score greater than 1 indicates that the rate of progress was higher than expected between the two evaluations (the patient made 12 months of progress during 6 months) . AccordiNevertheless, progress was observed after the initiation of auditory-verbal rehabilitation therapy. From the four evaluated domains, the personal-social and language areas show the lowest development estimate rate. We can conclude that language disorder can significantly influence the personal-social domain. These results are consistent with those obtained by Jareean Meizen-Derr et al. in a study which showed that language delays in hearing-impaired children have a direct impact on communication and the social field .The patient also presented developmental delay for both fine and gross motor abilities, which, after the initiation of physical therapy, have significantly improved.Another issue to be considered is brain plasticity in children with hearing loss and associated disabilities. Brain plasticity may be influenced by the etiology of hearing loss , and it may have effects on the therapeutic outcomes.The neurocognitive assessments need to establish a comprehensive framework of measuring outcomes in children with hearing loss, like auditory awareness, speech perception, language and speech development, neurocognitive development, and social skill assessments .The presented case reveals the importance of using the P1 CAEP biomarker in clinical practice along with a neuropsychological evaluation to assess the maturation of the central auditory pathways and to objectively quantify the results of auditory rehabilitation in children with hearing loss and associated disabilities.The authors confirm that there are no conflicts of interest.


The relevant information about the impacts caused by presence of emerging pollutants in mixtures on the ecological environment, especially on the more vulnerable compartments such as activated sludge (AS) is relatively limited. This study investigated the effect of ibuprofen (IBU) and triclosan (TCS), alone and in combination to the performance and enzymatic activity of AS bacterial community. The assays were carried out in a pilot AS reactor operating for two-weeks under continuous dosage of pollutants. The microbial activity was tracked by measuring oxygen uptake rate, esterase activity, oxidative stress and antioxidant enzyme activities. It was found that IBU and TCS had no acute toxic effects on reactor biomass concentration. TCS led to significant decrease of COD removal efficiency, which dropped from 90% to 35%. Continuous exposure to IBU, TCS and their mixtures increased the activities of glutathione s-transferase (GST) and esterase as a response to oxidative damage. A high increase in GST activity was associated with non-reversible toxic damage while peaks of esterase activity combined with moderate GST increase were attributed to an adaptive response. Pharmaceuticals and personal care products (PPCP) are one of the most relevant group of emerging pollutants because of their worldwide detection in practically all environmental compartments and their adverse biological effects –3. PPCP -1) concentrations of tetracycline on the microbial community of Sequent Batch Reactors and reported changes in microbial community and the proliferation of tetracycline-resistance genes [Several studies examined the influence of pharmaceuticals on biological wastewater treatment processes, mainly focusing on the effects of pollutants on the efficiency of plants for their removal, microbial growth and the rate of COD and nutrients removal –18. Arrice genes . Alteratce genes . In certce genes . Some stce genes . It has ce genes . Howeverce genes .In this work, we studied ibuprofen (IBU) and triclosan (TCS) as representative PPCP usually found in treated wastewaters , 27. IBUIn a previous work, we demonstrated the toxic effects of IBU and TCS, alone and in combination to the microbial activity of AS in terms of viability and respiration for short-time exposure (1h) and non-acclimated sludge by using a batch experimental design . Here, w12H7Cl3O2) and ibuprofen  both > 97% purity, were obtained from Sigma-Aldrich. The stock solutions of TCS and IBU were prepared in methanol and water, respectively and stored at 4 °C. Methanol in the testing solution was kept below 0.01% (v/v). Ultrapure water was generated from a Direct-Q™ 5 Ultrapure Water Systems from Millipore  with a specific resistance of 18.2 MΩ cm at 25 °C. Monochlorobimane (MCB), 2',7'-dichlorofluorescin diacetate (H2DCF-DA), reduced glutathione (GSH) and dimethyl sulfoxide  were purchased from Sigma-Aldrich. The components of synthetic feed were acquired from Conda-Pronadisa (Spain).Triclosan,  with a capacity to process 255,000 mei et al . The AS .8 g L-1 .-1, temperature at 20 ± 2 °C and pH at 8.0 ± 0.1, close to the pKa of TCS reported to be 8.1 [-1 range throughout all the experiments with minor deviations. The stability of pollutants under continuous bioassay conditions was verified by high pressure liquid chromatography (HPLC) before initiating the exposure experiments.The lab-scale AS systems used in this work were formed by three continuous aerated reactors and settlers, operated in parallel . The reao be 8.1 . Prior to be 8.1 . Acclima-1) and TCS (0.05 mg L-1) as well as their mixture (2 mg L-1 IBU + 0.05 mg L-1 TCS) in the influent. During the second period, starting day 8th, the dosing of pollutants doubled: 4 mg L-1 IBU, 0.10 mg L-1 TCS and for their mixture 4 mg L-1 IBU + 0.10 mg L-1 TCS. The amount of pollutants fed with the influent are listed in -1. Relative to the dry matter in AS, the pollutants were fed at rates of 125 mg kg-1 and 5000 mg kg-1 for TCS and IBU respectively during Period I and twice these figures for Period II. These figures were chosen to approach the upper estimates reported for the mass of PPCPs adsorbed or in general accumulated in the sewage sludge, which are 133 mg kg-1 and 2988 mg kg-1 for TCS and IBU respectively [For the exposure study, the bioreactors operated in parallel with individual pollutants and their mixtures. The bioreactors were monitored for 14 days, divided into two periods. The first 7 days they operated with continuous dosing of IBU , TOC, and ammonia-, nitrite-, and nitrate nitrogen were performed from the samples collected from the influent, effluent and sludge, as described in section 2.4. All assays were carried out in triplicate and the results were expressed as mean plus/minus standard deviation.The microbial activity was assayed by studying the oxygen uptake rate, esterase activity, oxidative stress and antioxidant enzyme activities. All measurements were carried out in triplicate and the results were expressed as mean standard deviation.-1).The specific oxygen uptake rate (SOUR) was determined as a measure of the whole aerobic biomass activity using the methodology developed elsewhere . Briefly™ FL, Ascent) at excitation and emission wavelengths of 485 and 520 nm, respectively. The incubation time for staining was 30 min at 25 °C.The fluorescein diacetate (FDA) hydrolysis technique was applied for evaluation of total esterase activity. FDA is a non-fluorescent molecule that diffuse into cells and are hydrolysed by intracellular non-specific esterases of bacteria . For thi2DCF-DA assay method according to the procedure reported in the literature [-1 H2DCF-DA for 30 min. The mixed liquor was transferred into 96-well microtiter plates  for fluorescence spectroscopy at excitation and emission wavelengths of 495 and 525 nm, respectively.The intracellular ROS in the sludge cells was measured by means of the Hterature . Prior t-1) were individually homogenized using an ultrasonic cell disintegrator Sonics-VibraCell  with a power density of 1 W/mL for 5 (net) min in intervals of 5 s with 5 s breaks to avoid sample heating [The enzyme-containing fractions from AS samples were obtained by sonication method. In brief, ice cooled samples of 10 mL sludge . The changes in DO due to H2O2 reduction were monitored and recorded. The results were expressed as O2 produced/mL of bacterial biomass.The method for determining the activity of CAT enzyme was based on the OT enzyme . The OxyT enzyme . BrieflyCell integrity and metabolic changes of microbial consortia in AS during pollutants exposure were also visually examined using confocal laser scanning microscopy (CLSM). Live/Dead BacLight Bacterial Viability kit  was used to evaluate bacterial viability in the sludge. In brief, this method differentiates viable and no-viability cells using Syto9, a fluorescent nucleic acid stain capable to penetrate cell membrane and bind DNA, and propidium iodide (PI), which is a fluorescent stain marking only membrane-damaged non-viable cells. The excitation/emission were 480/500 nm for Syto9 and 490/635 nm for PI. The micrographs were obtained in a Leica Microsystems Confocal SP5 fluorescence microscope. For this study, sludge samples (1 mL) were taken from the reactors before starting the exposure experiment: 0 days (Control), and at 7 days (Period I), and 14 days (Period II), in order to observe the effects of pollutants at the beginning and end of each period of exposure, respectively.COD was measured using spectrophotometer NOVA60 (Merck) after 2 h digestion. TOC values were determined by TOC-VCSH Total Organic Analyzer, Shimadzu. For the analysis of IBU and TCS, a solid phase extraction (SPE) was performed using, by 30 mg/3 mL Strata-X 33u Polymeric Reversed Phase cartridges (Phenomenex) as pre-concentration technique prior to quantitative determination. The extraction protocol was similar to that described elsewhere . IBU andA two-way ANOVA coupled with Tukey’s HSD (honestly significant difference) post-hoc test was performed for comparison of means. Statistically significant differences were considered to exist when p-value < 0.05. Results were provided as average and standard deviation.-1. In all cases, most of the remaining IBU and TCS were dissolved in the effluent with a minimum part adsorbed on sludge. The amount removed during the Period I represented 50 ± 6% for IBU and 95 ± 3% for TCS. During Period II, after 7 days of acclimation, the removal of IBU increased to 89 ± 4%. TCS also increased slightly to 98 ± 1%.In reactors treating binary mixtures of IBU and TCS, the removal efficiencies were not very different from reactors fed with IBU or TCS alone. The average removal for IBU during period I was 67 ± 7%, even higher than when IBU was fed alone. At the beginning of Period II the removal efficiency for IBU dropped until 20–30% in reactor operating with IBU+TCS mixture that in part recovered thereafter. For TCS, the removal was 89 ± 5% and 98 ± 1% (IBU+TCS reactors), only slightly lower than in reactors treating TCS alone. High removal efficiencies of anti-inflammatory drugs in aerobic treatment processes and low sorption potential of IBU on sludge have already been reported elsewhere . In our -1, TCS 0.05 mg L-1 or 2 mg L-1 IBU + 0.05 mg L-1 TCS) and Period II . The results showed a clear stimulation during the first 24–48 h after the introduction of pollutants, with maximum SOUR values even doubling the stationary state before exposure (~ 2 day-1). A peak in SOUR was not observed when doubling concentrations (Period II). During this period SOUR tended to decrease upon toxicant exposure. Our results showed that COD removal rate in acclimated reactors was in the 75–78% range  representing 1% and 5% respectively of their individual short-term EC50 values. A previous study performed according to the Standard Guideline OECD Test Guide 209 showed inhibitory effect of IBU and TCS to the respiration activity of AS measured in short-term (60 min) exposure [50 value for TCS was 0.32 ± 0.07 mg L-1 for a concentration of AS of 125 mg L−1, which represented 2560 mg kg sludge-1. The short-term EC50 for IBU was of 64 ± 13 mg L−1 or 512000 mg kg sludge-1 for the same conditions [In the current study the amount of IBU and TCS used for Period I were 125 mg kgexposure . The shonditions . The connditions .-1) TCS concentration upon sludge SOUR was observed before and attributed to increased microbial metabolic rates linked with uncoupling of oxidative phosphorylation [The effect of TCS to the microbial community function in anaerobic digesters has been described by McNamara et al. who found increased methane production and transient divergence of community structure in reactors treating relatively low (5–50 mg/kg) TCS concentration . TCS wasrylation . An initThe stimulation of microbial activity upon stress conditions has been observed in different stress circumstances. For example, increased salinity generally inhibits the bioactivity of sludge microorganisms, but the selective growth of microbes with higher tolerance to saline environment may lead to an increase in microbial respiration rates . SorptioIt is important to determine whether the impact of changes in toxicant concentrations on AS is reversible or not. Our results showed a possible warning situation in Period II (with higher concentration of IBU and TCS), at the beginning of which a sharp decrease in the removal of IBU was observed in reactors treating IBU+TCS mixture. At the end of Period II a clear decrease in COD removal was probably indicating a non-recoverable situation. Generally, the operators of WWTP take into account the quality of the effluent and the physical characteristics of the sludge to take measures on operational parameters. In the following section of this article we describe the results of a series of enzymatic activity tests in an attempt to provide a further insight into the mechanisms behind the stress conditions associated to emerging pollutants and their relationship with AS performance.50 values for the inhibition of esterase activity in activated exposed to IBU and TCS of 633 ± 63 mg L−1 sludge (125 mg L−1 of AS) and 1.94–5.34 mg L−1 (125–500 mg L−1 of AS) respectively [The effect of IBU, TCS and IBU+TCS to the enzymatic activity of AS is shown in ectively . The effectively . Esterasectively . The opeTypically, ROS are formed in cells through the reduction of oxygen by biological reducing agents such as NADH and NADPH, with the assistance of electron-transfer enzymes or through redox-active chemical species such as quinones and transition metals. Oxidative stress is a key cellular response in organisms exposed to environmental pollutants that takes place when the generation of ROS exceeds the capacity of antioxidant defenses. Low concentrations of ROS may facilitate signal transduction, enzyme activation and other cellular functions, but high concentration of ROS damages DNA, proteins or lipids and can lead to cellular transformations , 52. In Glutathione transferases are a group of eukaryotic and prokaryotic antioxidant enzymes that prevent the cellular damage caused by metabolically- and environmentally-produced ROS. GST transforms the reduced glutathione (GSH) used as a direct ROS scavenger to a variety of electrophilic compounds in a detoxification pathway. GST plays a crucial role in cell metabolism by protecting against oxidative damage and, therefore, GST has been used widely as a biomarker for assessing the toxic effects of pollutants that generate oxidative stress and moderate GST activity increase has been linked to detoxification routes upon pollutant exposure in bacteria . The effThe increase in GST and esterase activities was linked to the oxidative damage produced to AS microorganisms. A continuous increase in GST activity would be the fingerprint of a non-reversible damage that compromised bacterial viability and led to microbial decline and loss of bioreactor performance. Peaks of esterase activity combined with moderate GST increase would be associated to adaptive responses.-1 TCS and/or 2 mg L-1 IBU decreased CAT activity, which was probably the consequence of a damage in the antioxidant defense system that did not revert after acclimation.Finally, Moreover, there were significant differences (p < 0.05) between the concentrations and the control as well as the time exposure for the end-points assayed in this work. The differences between the concentrations in ROS and CAT measurements for IBU and TCS were not significant (p > 0.05).The results from fluorescence microscopy are depicted in Performance, oxygen respiration rate, viability and microbial enzymatic activity were investigated in AS reactors continuously exposed to IBU and TCS, alone and in combination, for 14 days, and the results related to the oxidative stress suffered by bacterial cells. IBU had no significant impact on reactor performance, while TCS led to significant decrease of COD removal efficiency. Toxic effects occurred upon continuous long-term dosages of the selected PPCPs. The toxicity to AS was caused by ROS generation and membrane integrity decline. Activity of key enzymes was affected by long-term exposure to IBU and TCS. Overall, our results showed for the first time that AS bacteria have the capacity to tolerate oxidative stress by activating their antioxidant system under continuous dosage of IBU and TCS.S1 File(DOCX)Click here for additional data file.


Inner ear hair cells are mechanosensory receptors that perceive mechanical sound and help to decode the sound in order to understand spoken language. Exposure to intense noise may result in the damage to the inner ear hair cells, causing noise-induced hearing loss (NIHL). Particularly, the outer hair cells are the first and the most affected cells in NIHL. After acoustic trauma, hair cells lose their structural integrity and initiate a self-deterioration process due to the oxidative stress. The activation of different cellular death pathways leads to complete hair cell death. This review specifically presents the current understanding of the mechanism exists behind the loss of inner ear hair cell in the auditory portion after noise-induced trauma. The article also explains the recent hair cell protection strategies to prevent the damage and restore hearing function in mammals. The inner ear is the most incredible and sophisticated organ of the body. It connects the personnel with the outer world in the form of hearing. The hearing loss is referred to as the most common sensory disorder that affects all age groups of the world population. The complex architecture of the mammalian organ of Corti makes it more susceptible to damage and is difficult to revert back into its native form , 2. AlthSensorineural hearing loss (SNHL) is referred to as the most common type of hearing disorder occurs due to the damage or loss of the hair cells, the neuron-hair cell synapses, and/or degeneration of neurons. The SNHL is not completely recoverable due to the lack of self-regenerative capacity of HCs and SGNs. The patients having SNHL may be provided with the hearing aids, and in case of severe to profound hearing loss, the patients have the only option of cochlear implants . HoweverThere are multiple etiologies of SNHL. At any age, the foremost reasons for hearing loss are genetic and the environmental factors. The main causes of SNHL are degenerative processes associated with aging, gene mutations, noise exposure, and the use of therapeutic drugs that have ototoxic side effects –16. InteStereociliary bundles found on the surface of hair cells are more susceptible to mechanical damage. The exposure to intense noise causes direct mechanical disruption of stereociliary structure and disrupts the normal cellular organization of the organ of Corti –24. Howeα (TNF-α) [The reactive oxygen species (ROS) are observed in the hair cells after the acoustic overexposure and exist there for about 10 days . The ROS (TNF-α) , 38.N-terminal kinase (JNK)/mitogen-activated protein kinase (MAPK) also induces a mitochondrial cell death pathway through the stimulation and translocation of Bax and procaspases, release of cytochrome c from mitochondria into the damage cell cytoplasm, and lastly, the cleavage of fodrin by activating caspases [α and interleukin 1β were upregulated by the microglia, fibrocytes, and neuronal cells at different time points in the noise-exposed cochlea [Two complex signaling pathways are commonly involved in noise-induced hair cell loss that are known as intrinsic and extrinsic cell death signaling pathways. In the cochlea exposed to intense noise, the extracellular stimuli initiate the extrinsic cell death signaling pathway by inducing the transmembrane death receptors. These receptors activate the caspase 8, which further triggers the distinct downstream signaling pathway leading to the activation of caspase 3 that mediates apoptosis in the outer hair cells . The intcaspases . A recen cochlea  suggestiα (TNF-α) pathway has also been activated in the noise-induced damage model [Caspase-independent apoptotic pathway is also involved in the hair cell loss. After the exposure to loud noise, the mitochondria participate in the apoptosis by releasing the apoptosis-inducing factors (AIFs) and endonuclease G (EndoG) through the outer mitochondrial membrane into the hair cell cytoplasm. EndoG translocates to cell nucleus in order to initiate apoptosis while the AIFs may not directly be involved in apoptosis but act as a redox factor in return to noise-induced oxidative stress . Howeverge model ; howeverge model .The acoustic overexposure results in an increase accumulation of free calcium ions in the outer hair cells that enter through the L-type calcium channels and cell intracellular stores such as the mitochondria and endoplasmic reticulum , 48. TheThese studies highlight the possible mechanisms of hair cell loss in a noise-induced cochlear damage model. Besides the fact that the oxidative stress and the activated death pathways after acoustic overexposure detrimentally affect the cochlea, it takes a longer duration to degenerate hair cells after acoustic injury, suggesting that there is a possibility to interrupt this diminishing process in the mammalian cochlea.Genetic factors may contribute to the development of noise-induced hearing loss. The individual humans and animals displayed a variation in the susceptibility to noise-induced damage even under controlled conditions. This difference in susceptibility may be influenced by the genetic factors. For the last two decades, several genetic studies were performed to identify the NIHL susceptibility genes and among them various NIHL susceptibility genes have been known to involved in different cellular pathways such as the genes involved in the potassium recycling pathway  , 52, oxiApart from this, recent studies also highlight that the synapse degeneration in the inner ear is another key contributor of NIHL. The synapsis between the inner hair cells and spiral ganglion neurons is more prone to cellular damage . GlutamaVarious strategies have been defined to protect inner ear hair cells from acoustic damage. Some of the useful external protective measures are to reduce the exposure of loud noise by eliminating the basic source of the noise. However, it is not possible to take this measure in all the conditions, particularly in the areas of high noise pollution such as in the industrial world. Therefore, the use of hearing protection devices is recommended and in somewhat effective to reduce the acoustic damage. Multiple studies have suggested the use of hearing protection devices that significantly reduce the loud noise exposure, thus minimizing the risk of hearing loss in the industrial workers –74. HoweBesides the external measures of hearing protection, the otoprotective treatment on a cellular level is mainly focused for the prevention of hair cell loss and induction of the self-repair mechanism to restore auditory function. The most effective strategies used to prevent hair cell loss after noise-induced damage are: (1) use of antioxidants, (2) inhibition of programmed cell death pathways, (3) anti-inflammatory therapies, and (4) neurotrophic factors.tert-butylnitrone (4-OHPBN) and N-acetyl-L-cysteine (NAC) after acoustic overexposure significantly reduces the noise-induced hearing loss [tert-butylnitrone and NAC) in combination that also protected the hair cells and afferent neurites from noise-induced damage and preserved the cochlear structural components [Antioxidants are the potential therapeutics used to protect inner ear hair cells from acoustic damage. The oral administration of antioxidant drugs such as 4-hydroxy-alpha-phenyl-ing loss . Anothermponents . In addimponents –81. Simimponents –84. The mponents –84. Howemponents . Kopke emponents . Collectmponents , coenzymmponents , resveramponents , glutathmponents , ginsengmponents , D-methimponents , and vitmponents –95. The mponents , 97. TheManipulation of intrinsic cell death cascades using different antiapoptotic inhibitors is also a promising strategy to protect hair cells after the noise-induced hearing loss. Multiple studies have shown the activation of MAPK/JNK pathways in cellular stress response. The blocking of this pathway using the JNK inhibitory molecules in the animal model provides significant protection against the acoustic trauma , 99. SimSeveral types of anti-inflammatory drugs have been reported to rescue the hearing deterioration in the inner ear induced by the sound overexposure. Particularly, the use of steroids such as dexamethasone and dehydroepiandrosterone reduces the noise-induced trauma in the guinea pig and mice models –107. TheAlthough the neurotrophins are the key regulators for differentiation, survival, and maintenance of neuronal cells, several studies have reported their otoprotective role against the noise-induced hearing loss –113. NeuIt is very fascinating to observe that several strategies and drugs have been discovered to protect the inner ear hair cells from acoustic damage. A common strategy in recent years appears to target and manipulate the programmed cell death pathways and involves the use of antioxidants to control the oxidative stress in hair cells. After noise-induced ototoxic damage, there are different signaling pathways activated in the cochlea that induce hair cell death. The interruption in one of these death signaling cascades by specific inhibitor might not be that effective to rescue the auditory function until the multiple drugs or molecular inhibitors are not used in combination. The cellular cascades are interlinked with each other, and there is a possibility that the inhibition of one pathway with specific inhibitor might result in the activation of other cell death pathways in hair cells. Thus, a synergistic approach would be more beneficial to restore the hearing loss.As reviewed above, to study the noise-induced hearing loss in the animal model, the researchers used various approaches such as different animal species, sound intensity, frequency spectrum, and continuous or impulse noise. This sometime creates a conflict among the results of some drug studies that have found to be otoprotective by one group while reported as ineffective by the other group. The most probable reason for these conflicts is the difference between genetic backgrounds of different animal species that have different sensitivities to intense sound; thus, there is a possibility for different responses of same drug in different animal models. It is important to understand that the majority of animal studies showed a statistically significant impact when the protective drug reduces the hearing loss by 2-3 decibels (dB). However, in humans, this shift should be more than 10 dB to be effective for the auditory performance, and the hearing below this level may have a very less impact on person hearing capability. Therefore, in the future, it is important to first perform the otoprotective therapeutic experiments in different animal models with different acoustic exposure conditions before taking it into the clinical trials.In recent years, research on the noise-induced hearing loss is focused in order to develop various therapeutic strategies for appropriate protection of hair cells from any damage and to restore the auditory function after acoustic trauma. The momentum builts up by these studies on the effectiveness of different otoprotective agents such as the antioxidants, anti-inflammatory agents, and neurotrophic factors, and manipulation of the intrinsic cell death pathways in hair cells will likely drive the complete development of therapeutic interventions for restoring the noise-induced hearing loss in the future. However, identification of the optimal conditions such as the dose regimen, effective route of administration, and timings for new drugs and their synergistic plans to treat sound-induced hearing loss in patients is important to be focused in the future experiments.


Hearing loss, the most common neurological disorder and the fourth leading cause of years lived with disability, can have profound effects on quality of life. The impact of this “invisible disability,” with significant consequences, economic and personal, is most substantial in low- and middle-income countries, where >80% of affected people live. Given the importance of hearing for communication, enjoyment, and safety, with up to 500 million affected globally at a cost of nearly $800 billion/year, research on new approaches toward prevention and treatment is attracting increased attention. The consequences of noise pollution are largely preventable, but irreversible hearing loss can result from aging, disease, or drug side effects. Once damage occurs, treatment relies on hearing aids and cochlear implants. Preventing, delaying, or reducing some degree of hearing loss may be possible by avoiding excessive noise and addressing major contributory factors such as cardiovascular risk. However, given the magnitude of the problem, these interventions alone are unlikely to be sufficient. Recent advances in understanding principal mechanisms that govern hearing function, together with new drug discovery paradigms designed to identify efficacious therapies, bode well for pharmaceutical intervention. This review surveys various causes of loss of auditory function and discusses potential neurological underpinnings, including mitochondrial dysfunction. Mitochondria mitigate cell protection, survival, and function and may succumb to cumulative degradation of energy production and performance; the end result is cell death. Energy-demanding neurons and vestibulocochlear hair cells are vulnerable to mitochondrial dysfunction, and hearing impairment and deafness are characteristic of neurodegenerative mitochondrial disease phenotypes. Beyond acting as cellular powerhouses, mitochondria regulate immune responses to infections, and studies of this phenomenon have aided in identifying nuclear factor kappa B and nuclear factor erythroid 2-related factor 2/antioxidant response element signaling as targets for discovery of otologic drugs, respectively, suppressing or upregulating these pathways. Treatment with free radical scavenging antioxidants is one therapeutic approach, with lipoic acid and corresponding carnitine esters exhibiting improved biodistribution and other features showing promise. These compounds are also histone deacetylase (HDAC) inhibitors, adding epigenetic modulation to the mechanistic milieu through which they act. These data suggest that new drugs targeting mitochondrial dysfunction and modulating epigenetic pathways via HDAC inhibition or other mechanisms hold great promise. It enriches our lives with the sound of music and alerts us to imminent danger that can be heard although perhaps not seen. The Greek physician-philosopher Alcmaeon of Croton2–5 and his two protagonist followers, Praxagoras of Kos6 and the great Alexandrian physician of Chalcedon,7 Herophilus,3 propounded that hearing is a construct of the brain, where external sound channeled to it through the ears  is interpreted.6Vision is the pre-eminent sensory means by which we navigate the world around us,8 The World Health Organization (WHO) estimates that the annual cost of unaddressed hearing loss is in the range of $750–790 billion globally.9 In the “Global Burden of Disease,” impaired hearing represents the fourth leading cause of disability worldwide.10–12 About 5–7% of the world's population (∼360–500 million people) has a hearing disability13 that is severe enough in more than 80% of people older than 85 years to interfere with their ability to communicate effectively.10 In children, impaired hearing can impinge on their academic potential and social development13 with lifelong adverse consequences.14 Hearing loss due to mechanical (conductive) and/or anatomical issues in the outer and/or middle ear , one of the most common types of SNHL.18 Noise pollution is a growing health problem around the world.19–22 For example, in the United States alone, it is estimated that more than 25% of the adult population has measurable hearing loss caused by exposure to harmful noise.10 Irreversible hearing loss can also be the product of disease24 and is often an unfortunate side effect of the aminoglycoside antibiotics25–30 and platin-based anticancer drugs.30–36 Regrettably, once damage has occurred, hearing aids and cochlear implantation are the only compensatory options presently available for affected individuals.37Although the array of techniques used in the diagnosis of sensorineural hearing loss  is progressively being expanded and refined,39 SNHL results from damage to the organ of Corti  and cognitive decline, cognitive impairment, and dementia—suggesting that ARHL may be a relevant biomarker and a targetable modifiable risk factor for dementia.43–46 However, given that advanced age itself is a leading risk factor for dementia,47–49 the meta-analysis correlation is not surprising.There is a substantial and growing worldwide unmet medical need for a pharmaceutical approach to treating hearing impairment.of Corti , causing50–52  and, over time, they succumb to an increasing cumulative degradation of their cellular energy production and performance—driving the cell toward death (apoptosis) and/or premature senescence .53–59 Energy-demanding cells such as neurons and the vestibulocochlear hair cells , as well as in the nuclear genome , are known to cause hearing deficits .24,62,6373–76 Often overlooked among the many causes of impaired hearing, including deafness, is virus-induced hearing loss.77–79 Although the mechanisms of hearing loss/deafness associated with viral infections remain largely undefined,80 viral infections activate a cascade of mitochondrial antiviral innate immune responses that include nuclear factor kappa B (NF-κB)81 and nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) signaling pathways.76In addition, mitochondria are key regulators of our innate and adaptive immune responses to viral infections.83 While a full understanding of these pathways awaits further study, it seems clear that epigenetics plays a significant role. Mammalian vestibulocochlear hair cells are a stress-sensitive, nonregenerative cell type and, like the retinal cells of the eye,1 are not replaced when they are injured or die.84–86 Interestingly, emerging research indicates we selectively amplify directional sound in a noisy environment by unconsciously utilizing eye–ear coordination to integrate visual cues with the auditory information.88 Assessing vision and oculomotor function is essential in the diagnostic evaluation of vestibulocochlear auditory impairments,89 particularly in patients with idiopathic etiology.91Unmitigated oxidative stress  and epigenetically altered expression of genes sensing oxidative stress are significant contributors to the pathogenicity of neurodegenerative disorders .92–94 and regenerative medicine are making inroads toward regenerating hair cells with the aim of reversing hearing loss.95 Some of the achievements demonstrate therapeutic potential,86 but a clinical application is still a long way off.39 In the more immediate future, shielding hair cells from oxidative damage and/or rescuing injured hair cells from falling into apoptosis by pharmacological treatment with free radical scavenging antioxidant compounds portend a promising therapeutic approach.96–99Impressive advances in gene therapyNrf2/ARE gene expression pathways and/or suppressing NF-κB signaling are cogent targets for pharmaceutical intervention strategies.34 Many natural and synthetic compounds are known inhibitors of NF-κB signaling100—butyric acid (butyrate)101–105 and α-lipoic acid (5-pentanoic acid)106–110  with respect to the proapoptotic protein, Bcl-2-associated X protein (BAX) in a BAX/Bcl-2 ratio of 1:2.110–113 Again, note the implied potential of drugs that modulate epigenetic pathways.Upregulating )106–110  are amonPMX500FI , also ac114–119 acting on signaling mechanisms through both receptor-mediated pathways and nonreceptor-mediated antioxidant processes in a variety of cell types120—including cochlear hair cells.122 In humans, ALA is a functionally versatile endogenous molecule enzymatically synthesized in mitochondria from octanoic acid.123 It is a key cofactor in the construction of vital metabolic multienzyme complexes, including pyruvate dehydrogenase and the glycine cleavage system.123 It is also a strong antioxidant125 and anti-inflammatory125 agent capable of activating and modulating signal transduction pathways,126 upregulating the expression of nerve growth factor, and augmenting the conduction velocity of motor nerves.127ALA has been extensively researched as a neuroprotectant,128 ALA is a potent activator of Nrf2, a transcription factor encoded by NFE2L2 that helps regulate cellular redox balance and protective antioxidant and phase II detoxification responses in mammals.50 Dietary antioxidant supplements are commonly sought by patients and caregivers for treating primary mitochondrial disorders.65 The role of antioxidants in prevention of age-related hearing loss has been reviewed by Tavanai and Mohammadkhani.129 In one of the reviewed studies, C57BL/6 mice fed with control diet or diet containing 1 of 17 antioxidant compounds , ALA, carotene, carnosine, coenzyme Q10, curcumin, tocopherol, epigallocatechin-3-gallate, gallic acid, lutein, lycopene, melatonin, proanthocyanidin, quercetin, resveratrol, or tannic acid), ARHL was nearly completely prevented by ALA and coenzyme Q10 and partially by NAC, but not by the other compounds.130 Unfortunately, this strategy showed no significant benefit in an interventional human study.131The expression of ∼1% (∼200–250 genes) of the protein-coding human genome is modulated in concert with the Nrf2/ARE signaling pathway.131 study may not truly address the ability of antioxidants to prevent ARHL because the design of the study was not directed toward prevention, and damaged cochlear hair cells are not restored by antioxidants.129 In studies aimed at preventing hearing loss in aged animals, ALA was shown to confer significant hearing preservation.108 Similar results between human and animal studies99 were also observed with the use of l-carnitine—an endogenously synthesized molecule mostly obtained from the diet.65However, the results from the Polanski and Cruz105 Sodium butyrate is a well-documented HDAC inhibitor105 that has demonstrated anti-inflammatory NF-κB inhibition properties.101–105 Butyrate mediates NF-κB activation by rescuing the redox machinery and controlling reactive oxygen species105 that are highly injurious to hair cells132 by suppressing the NF-κB signaling pathways.105NF-κB is a transcription factor that regulates the expression of a variety of genes involved in inflammation and immunity.50 Furthermore, a recent Phase I clinical trial in age-related macular degeneration evaluating the safety and tolerability of ALA in 15 subjects, 65 years of age or older, showed that high doses (800–1200 mg) of racemic ALA cannot be tolerated very well by patients.133 Thus, in the treatment of hearing loss, a need for ALA and butyrate derivatives having more clinically suitable pharmacokinetics is a challenging pharmaceutical objective.Although ALA and butyrate are common food and diet supplements that can be safely taken in high doses, their bioavailability is not prolonged or sustained at an effective therapeutic level.135 Finally, readers are directed to Hearing impairment is a major global health concern; its massive impact seemingly unrecognized until recently, and the affected population largely untreated. Preventing, or at least delaying or reducing, some hearing loss may be possible by avoiding excessive noise exposure and addressing contributory factors such as cardiovascular risk, infectious diseases, neurological disorders, and drug toxicity. However, these interventions will not be sufficient given the sheer magnitude of the problem. Thus, in view of recent advances in our understanding of the underlying mechanistic pathways—both mitochondrial and epigenetic—that govern hearing function, coupled with new drug discovery paradigms that can today be exploited to identify new and effective therapies, the time is ripe to tackle hearing loss with novel medicines. Alcmaeon of Croton remarked that vision and hearing are constructs of the brain. We see and hear in our dreams and in some aspects of disease conditions, such as high fever, schizophrenia, psychosis, or the later stages of dementia, and our dreams may blend into our conscious state immersed in auditory and/or visual hallucinations and delusions. The most common hallucination in schizophrenia is hearing voices.