Mnemiopsis leidyi. We applied tests of phylogenetic incongruence to identify nine genes that were likely transferred horizontally early in ctenophore evolution from bacteria and non-metazoan eukaryotes. All but one of these HGTs (an uncharacterized protein) are homologous to characterized enzymes, supporting previous observations that genes encoding enzymes are more likely to be retained after HGT events. We found that the majority of these nine horizontally transferred genes were expressed during development, suggesting that they are active and play a role in the biology of M. leidyi. This is the first report of HGT in ctenophores, and contributes to an ever-growing literature on the prevalence of genetic information flowing between non-animals and animals.Horizontal gene transfer (HGT) has had major impacts on the biology of a wide range of organisms from antibiotic resistance in bacteria to adaptations to herbivory in arthropods. A growing body of literature shows that HGT between non-animals and animals is more commonplace than previously thought. In this study, we present a thorough investigation of HGT in the ctenophore  Evolution is commonly thought to occur by descent with modification from a single lineage. However, evidence has shown that genomes from bacteria, archaea, and eukaryotes are typically chimeric, resulting from horizontal  gene transfers . As suchAlthough HGT is generally accepted as an important evolutionary mechanism in prokaryotes , it remabona fide HGTs, as “animal” sequences will appear among the top BLAST hits for a particular HGT, leading to false negatives  are marine invertebrates that are morphologically characterized by eight rows of cilia used for movement. They typically live in the water column, but the group includes benthic species as well . PhylogeMnemiopsis leidyi. Our process includes identification of HGT candidates by alien index and confirmation by phylogenetic hypothesis testing to provide statistical support in an evolutionary framework. Furthermore, we analyze gene expression profiles during development to obtain clues as to the function of these HGTs in M. leidyi.Here, we apply a rigorous framework to identify and confirm HGTs in the ctenophore M. leidyi gene models (ML2.2) , archaea (2), non-animal eukaryotes (5), and animals (12). See http://ryanlab.whitney.ufl.edu/downloads/alien_index/ for the entire list of taxa. HGT index values were computed by the difference in the highest non-animal and animal bit scores generated from the alien_index database. The alien_index program is available at: https://github.com/josephryan/alien_index.As part of this project, we developed the program alien_index and complimentary metazoan/non-metazoan sequence databases to automate the generation of alien index  and HGT  (ML2.2)  against E-value cutoff of 0.1. We included only the first sequence if there were hits to sequences from species in the same genus  from each of the following fully sequenced animals from version 0.01 of the alien_index database: Amphimedon queenslandica, Trichoplax adhaerens, Nematostella vectensis, Capitella teleta, Drosophila melanogaster, and Homo sapiens. Sequences were aligned against the corresponding putative HGT using MAFFT , preventing us from performing additional phylogenetic analyses on these sequences. We considered the lack of animal BLASTP hits below our cutoff as sufficient evidence that these six were clearly HGTs. ML018031a and ML00882a only had two BLASTP hits to animal sequences. Since it was unclear if this resulted from contamination, we were unable to test these genes using phylogenetic approaches, so they were removed from contention as HGTs.We applied a phylogenetic approach to confirm putative HGTs. HGT candidates from alien_index were used as queries for BLASTP against NCBI’s RefSeq database  using thme genus . We alsong MAFFT  and trimng MAFFT  , we performed multiple AU analyses using bootstrap trees as suboptimal trees (similar to https://github.com/josephryan/2018-Hernandez_and_Ryan_HGT).We explicitly tested topologies in opposition to HGT  with the SOWH test using SOWHAT  and the M. leidyi genome browser  against the transcriptome of an M. leidyi individual collected from St. Augustine, FL, USA, as well as seven other ctenophore transcriptomes reported in Bolinopsis infundibulum, Beroe abyssicola, Dryodora glandiformis, Pleurobrachia bachei, V. multiformis, Coeloplana astericola, Euplokamis dunlapae. For these searches we used default parameters and an E-value cutoff of 0.1.We verified that HGT candidates were not the result of bacterial contaminants by using the  browser  to examiM. leidyi was recently generated from single-embryos over the first 20 h of embryogenesis (GSE70185), as well as additional time points for M. leidyi generated after this publication (GSE111748).An extensive transcriptomic developmental timecourse of ogenesis . To examM. leidyi gene models (ML2.2) using bowtie2 version 2.2.3 . These additional data were produced by the same researchers  from the original study using the same methods and facilities. To create a baseline for what is considered adequate expression during development, we summed median transcripts per million (tpm) values for all replicates along the 25 time points for each of our nine confirmed HGTs. HGTs that had summed median read counts of 100 or greater were classified as being expressed sufficiently to have roles in development. We chose a value that was 10 times stricter than the minimum criteria in Since the publication of E-value ≤ 0.1) were assigned to HGT candidates. We also associated HGTs with Pfam-A domains using the MGP Portal under the Mnemiopsis Gene Wiki  as absent from all other animals. We analyze these six further below.-animals . We iden-animals . In addi-animals . All butA. queenslandica, T. adhaerens, N. vectensis, C. teleta, D. melanogaster, and H. sapiens that were top hits from our initial BLASTs of the alien_index database (p < 0.05) from the metazoan-constraint trees in both the SOWH and AU analyses . If ctenophores are the sister group to the rest of animals, vertical inheritance remains a possibility for these cases. As such, these tests support a total of nine HGTs.We then analyzed the BLAST results of the seven HGT candidates confirmed by phylogenetic analyses and the six genes which were absent in other animals . We removed four of these genes from contention  because the top BLAST hits against RefSeq were either Choanoflagellatea or Filasterea  . Eight HGTs were found on scaffolds with intron-containing genes and eight HGTs contained introns . We also performed reciprocal best BLASTP searches for each of the nine HGTs against seven of the ctenophore transcriptomes published in B. infundibulum, B. abyssicola, D. glandiformis, P. bachei, V. multiformis, C. astericola, E. dunlapae. Each HGT was present in the transcriptome of at least one other ctenophore species and in the Florida M. leidyi transcriptome  from single-embryo RNA-Seq analyses over 20 h for each of the nine confirmed HGTs to identify those HGTs that were expressed during development. Six of the nine HGTs had sums greater than 100 , suggestWe used phylogenetic evidence to determine the origin of these nine HGTs. Four HGTs originated from bacteria and five from non-animal eukaryotes . We founM. leidyi individuals, as well as in many other ctenophore species  genes . We conf species . Six HGTvolution . This deProteobacteria is the most abundant group of bacteria associated with ctenophores  rather than informational genes . InformaIdentifying HGTs can be challenging due to bacterial associations with hosts , DNA extTo overcome some of these challenges, we developed alien_index to automate the generation of alien index and HGT index scores for rapid identification of HGT candidates. We confirmed HGTs by using rigorous phylogenetic approaches to address the problems associated with the lack of evolutionary perspective from BLAST methods. Our phylogenetic tests of incongruence provided clear metrics from which to judge the level of certainty applied to each HGT candidate. Our study showed that many of the predictions based on BLAST did not stand up to hypothesis testing and suggest that the similarity between sequences that cause high alien indices do not necessarily provide true phylogenetic signal. Consequently, incorporation of phylogenetic likelihood-based methods are necessary when performing HGT analyses in animals.M. leidyi. However, many more studies will be necessary to gain a comprehensive overview of HGT and the mechanisms by which HGT occurs in animals.The importance of HGT as an evolutionary mechanism in prokaryotes and eukaryotes has been underestimated. While studies of HGT in animals are gradually becoming more accepted, many challenges remain to quantify the extent of HGT and its impacts. To mitigate some of these challenges, rigorous approaches that employ both BLAST- and phylogenetic likelihood-based methods should be applied to future HGT studies in animals. Here we provided evidence of nine cases of HGT in ctenophores by applying these rigorous methods (among others), and found similar patterns of transfer between prokaryotes and eukaryotes with preference for operational genes. It should be noted that we implemented an extremely conservative approach and there are likely to be more HGTs in 10.7717/peerj.5067/supp-1Supplemental Information 1Click here for additional data file.


Ganoderma lucidum. They have been investigated for their biological activities, including antibacterial, antiviral, antitumor, anti-HIV-1, antioxidation, and cholesterol reduction functions. Inducer supplementation is viewed as a promising technology for the production of GAs. This study found that supplementation with sodium acetate (4 mM) significantly increased the GAs content of fruiting bodies by 28.63% compared to the control. In order to explore the mechanism of ganoderic acid accumulation, the transcriptional responses of key GAs biosynthetic genes, including the acetyl coenzyme A synthase gene, and the expression levels of genes involved in calcineurin signaling and acetyl-CoA content have been analyzed. The results showed that the expression of three key GAs biosynthetic genes  were significantly up-regulated. Analysis indicated that the acetate ion increased the expression of genes related to acetic acid assimilation and increased GAs biosynthesis, thereby resulting in the accumulation of GAs. Further investigation of the expression levels of genes involved in calcineurin signaling revealed that Na+ supplementation and the consequent exchange of Na+/Ca2+ induced GAs biosynthesis. Overall, this study indicates a feasible new approach of utilizing sodium acetate elicitation for the enhanced production of valuable GAs content in G. lucidum, and also provided the primary mechanism of GAs accumulation.Ganoderic acids (GAs) are a type of highly oxygenated lanostane-type triterpenoids that are responsible for the pharmacological activities of  Ganoderma lucidum, also known as ‘the mushroom of immortality’ and ‘the symbol of traditional Chinese medicine’, is one of the best-known medicinal macrofungi in the world [G. lucidum [he world ,2. Ganod lucidum . Modern  lucidum ,5,6.GAs are synthesized via the mevalonate pathway (MVA), wherein acetyl-CoA is converted to 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) through a series of chemical reactions, and further to mevalonate (MVA) to isopentenyl pyrophosphate (IPP) to farnesyl diphosphate (FPP) to squalene, and finally to lanosterol ,8,9. TheGanoderma triterpenoids have been widely recognized, their sustainable production has become a necessity in order to fulfill the increasing demand. Various methods aimed at increasing the yields of GAs have been reported. For example, during the vegetative mycelia stage, considerable efforts have been made to improve GAs production in the submerged cultivation of G. lucidum, such as the optimization of cultivation conditions [G. lucidum. In general, the content of GAs in the fruiting body was significantly higher than that in the mycelia. However, it remains unclear whether the methods reported to increase GAs production in the mycelia stage could also be applied toward the fruiting body stage.As the pharmacological activities of nditions , the addnditions ,18,19, anditions ,21. SignackA and pta, respectively) or acetyl-CoA synthetase  [acs and then regulate intracellular metabolism and gene expression [+), the main channel ion, may have similar impacts on metabolite production. A high level of extracellular Na+ has been reported to increase intracellular Ca2+ to cope with salt stress in yeast [2+ signals [+ has been observed with respect to alatoxin [Volvariella volvacea, the mean weight increased when sodium acetate was applied as a spray during cultivation [G. lucidum.After the potential for acetate to be used as a fungal feedstock was discovered, efforts have been made to produce various biochemicals from acetate based on an understanding of its metabolism . Extrace by acs) . Acetic pression . Channelpression . Sodium in yeast  and ricein yeast  by trigg signals . A similalatoxin  and GAs alatoxin . In Volvtivation . TherefoG. lucidum using sodium acetate as an inducer. To explore the underlying mechanisms responsible for its accumulation, the transcripts of genes encoding key enzymes in the GAs biosynthetic pathway, acs and those of genes in the calcineurin signal pathway, were also assayed.The present study aimed to enhance GAs accumulation in the fruiting body of p < 0.05) in the dry weights of the single fruiting bodies between the treatments and the control groups. The average dry weight of a single fruiting body was 8.96% to 16.64% higher than the control in the cases of sodium acetate and acetic acid treatments, respectively (Volvariella volvacea [The results showed that there was significant difference (ectively . Howeverectively . These ovolvacea .p < 0.01)  . In the hmgs, fps, and sqs were higher than the control group by folds of 32.8, 6.9, and 12.0, respectively (p < 0.05). In the acetic acid treatment, the transcription level of hmgs was 4.9-fold higher (p < 0.05) than the control group. In treatment process, there were three key GA biosynthetic genes  that were up-regulated under the influence of sodium acetate, while only one gene (hmgs) was up-regulated in the acetate acid condition. The results of qRT-PCR were consistent with the variation of GAs content in G. lucidum, leading to an increased accumulation of GAs.In acs and measured the acetyl-CoA content in fruiting bodies of G. lucidum treated with sodium acetate or acetic acid. Under sodium acetate treatments, the transcription levels of homologous acs  were 6.96, 5.12, and 4.26-fold that of the control, respectively. In the acetic acid treatment, the transcription levels of homologous acs  were 4.32, 4.35, and 5.46-folds that of the control, respectively  were quantified  was reported to induce the biosynthesis of GAs in the mycelium of  lucidum . Recentl lucidum . GAs areG. lucidum. However, the mechanism by which sodium acetate increased GAs biosynthesis largely remains unknown.A recent study found that spraying SA during the fruiting body formation enhanced the accumulation of GAs. SA increased the triterpenoid content by 23.32% compared to the control [acs, and acetyl-CoA is used in many metabolic pathways [hmgs, fps, and sqs were highest in the sodium acetate treatment. Presumably, the higher transcript levels of GAs biosynthetic genes resulted in enhanced GAs accumulation, which is in agreement with what has been reported in other studies on the biosynthesis of GAs [+ in inducing the biosynthesis of GAs. Here, we found that the transcription levels of genes encoding Ca2+/Na+ exchange and the genes in the calcineurin signal pathway were up-regulated in the sodium acetate condition. It indicates that while acetate ions directly enhance the biosynthesis of GAs, Na+ ions regulate the process of GAs biosynthesis.In order to survey the mechanisms by which sodium acetate increased the biosynthesis of GAs, we have explored the content of acetyl-CoA and gene expression levels in acetyl-CoA assimilation and the calcineurin signal pathway. The highest value of acetyl-CoA content, 398.52nmol/g, was recorded in the acetic acid treatment in pathways . Howevers of GAs . This maacs and acetyl-CoA assimilation, thereby increasing GAs biosynthesis. On the other hand, Na+ induction would be expected to enhance cytosolic Ca2+ to induce GAs biosynthesis through the calcineurin signal pathway to up-regulate its biosynthetic genes at the transcriptional level  was obtained from the Agricultural Culture Collection of China. It was grown at 28 °C in potato dextrose agar medium for 7 days. Spawn was prepared in polypropylene bags (18 cm in width × 36 cm in length). The growing medium was sawdust (78%), mixed with 20% wheat bran and 2% gypsum based on dry weight, and subsequently adjusted to a moisture content of 65%. The bags were filled with moistened substrate, sterilized at 121 °C for 2 h, and subsequently allowed to cool to room temperature. The inoculated substrate was incubated at 28 °C for mycelial colonization. The air temperature in the cultivation room was maintained at 28 °C and 80–85% relative humidity. The bags were sprayed with 20 mL of either sodium acetate (4 mM) or acetic acid (4 mM) at regular intervals of 4 days for a total period of 40 days. Control bags were sprayed with distilled water. When the white margins of the pileus disappeared, mature fruiting bodies were collected from the bags, weighed, and dried at 60 °C to a constant weight.g for 10 min, one milliliter of the supernatant was obtained and mixed with 2 mL of 5% (w/v) vanillin and 5 mL of perchloric acid. The mixture was maintained in a water bath at 60 °C for 20 min, cooled in ice water for 10 min, and then followed by adding 200 μL of glacial acetic acid in 32 μL of the mixture. The total GAs content was determined based on a colorimetric reaction involving vanillin-perchloric acid and glacial acetic acid with GAs. The absorbance was measured at 550 nm with a spectrophotometer using ursolic acid as a reference.Dried fruiting bodies were ground to a fine powder. Total GAs content were extracted from each sample separately by adding 1 g of grounded powder into 95% of ethanol (50 mL) at a frequency of 70 kHz in an ultrasonic bath for 2 h. After centrifugation at 4000× G. lucidum using RNAiso Plus (TaKaRa), and then treated with RNase-free DNaseI (TaKaRa). Synthesis of cDNA was with a TransStart All-in-One first-strand cDNA synthesis supermix for quantitative real-time PCR (qRT-PCR) according to the manufacturer’s protocol. The transcript levels of hydroxymethylglutaryl-CoA synthase (hmgs), hydroxymethylglutaryl-CoA reductase (hmgr), farnesyl pyrophosphate synthase (fps), squalene synthase (sqs), oxidosqualene cyclase (osc), and sensor genes , encoding Na+-ATPase (ena1) and Ca2+-ATPase, were determined by qRT-PCR based on a relative method using a LightCycler® 96 SW 1.1 with SYBR Green. Post-qRT-PCR calculations analyzing relative gene expression levels were performed according to the 2−∆∆Ct method [G. lucidum 18S rRNA gene in control samples. For each gene, the expression level of the reference sample was set as 1.0, and the transcript level was measured as the increase in its mRNA level over that of the reference sample. The primer sets were used as previously described and are listed in acs homolog genes  in the G. lucidum genome [Total RNA was extracted from fruiting body samples of t method . The geng for 10 min. Acetyl-CoA content was then measured at 340 nm, using a spectrophotometer as described by the protocol.Acetyl-CoA in fruiting bodies was extracted and measured using an acetyl-CoA assay kit . Briefly, fruiting body samples (100 mg) were ground in a mortar in an ice bath according to the manufacturer’s protocol, and then centrifuged at 8000× A completely randomized design with 30 replicates was used. All the data are expressed as the mean ± standard deviation (SD). Data were subjected to analysis of variance using the Student’s t-test, and the mean values indicating statistical significance were compared by Duncan’s multiple-range test using SPSS 17.0 software.G. lucidum. In the present study, we have investigated the inducing effect on GAs biosynthesis during fruiting body formation using sodium acetate. The results showed that sodium acetate not only enhances the GAs content, but also can increase the yield of fruiting body in G. luncidum. This study represents an important stimulus for future research on the biosynthesis and cultivation of GAs in the medicinal fungi G. luncidum.GAs are an important secondary metabolite that display mainly active content in


Beak treatment of commercial laying hens remains an important management practice as it is one of the most effective methods of controlling and preventing cannibalism. Infrared beak treatment is the most recent beak treatment method to be utilized and the available literature shows that it has less of a negative impact on birds compared to older methods of beak treatment. Although there is considerable research evaluating the impact of infrared beak treatment on the production and welfare of laying hens, it is still not fully understood how it affects the beak tissue during the first few days post treatment. This is important to understand as it can provide insight into whether or not treated birds are experiencing pain, which has consequences for both welfare and productivity. This study examined the effect of infrared beak treatment on the histology of the beak during early life (first 21 days). Epithelial regeneration started as soon as five days post treatment. There was no evidence that infrared beak treatment resulted in the formation of neuromas or any other indication of chronic pain.Infrared beak treatment has less of a negative impact on laying hen welfare compared to other methods of beak treatment; however, it is still not fully understood how infrared beak treatment affects the beak tissue during the first few days post treatment. The objective of this study was to examine the histology of infrared beak treated vs. untreated beaks of 2 strains of layer chicks during early life. One-hundred Lohmann Brown-Lite (LB) and 100 Lohmann LSL-Lite (LW) chicks were obtained; 50 chicks per strain were infrared beak treated post hatch (IR) with the remainder being sham untreated controls (C). Data collected included presence of beak sloughing, length, and histology. Histology slides were analyzed and scored on a scale of 0 to 4, with 0 indicating no lesions and 4 indicating severe inflammation. Sloughing of the treated beak tissue began at 10 days and was complete by 20 days. IR pullets had shorter beak lengths once sloughing was initiated and less overall beak growth. No differences in healing scores were found between treated LB and LW beaks; all treated LB beaks were healed by 21 days while some LW beaks still showed inflammation. Overall, infrared beak treatment was effective at reducing beak growth post treatment. Healing occurred post treatment in both strains as evident by complete regeneration of the epithelium and a reduction in inflammation. In commercial egg production, hens are beak treated to control cannibalism and feather pecking. Traditional methods of beak treatment, such as hot-blade trimming, involve the use of a heated blade to cut and cauterize the beak tissue . Hot-blaAs with many other animal husbandry practices, beak treatment often raises concerns  regarding its effect on welfare. Various alternatives to beak treatment, such as varying light intensity, using low feather pecking strains, and the use of enrichments, have been proposed and studied . HoweverThe majority of the scientific literature on welfare and beak treatment has focused on hot-blade trimming, and often these results are extrapolated to what might occur with the use of infrared beak treatment. However, previous studies comparing hot-blade trimming and infrared beak treatment have found that infrared beak treatment represents a more welfare-friendly method of beak treatment because it has less of a negative effect on pullet and hen production and welfare ,7,8. InfThere is still limited research investigating the long-term effects of infrared beak treatment on the neurophysiology and anatomy of the beak. Chicks that were treated using infrared beak treatment at 1 day of age were found to have neuromas, which were observed at 32 days of age and persisted into adulthood . NeuromaDespite the research that has been conducted on the impact of infrared beak treatment on the production and welfare of egg production pullets, gaps in the scientific literature regarding the effects of infrared beak treatment on beak histology and healing still exist. Therefore, the objectives of this study were to understand the histology of infrared beak treated versus untreated beaks during early life and investigate how infrared beak treatment affects the beak length and healing response of Lohmann Brown-Lite (LB) and Lohmann LSL-Lite (LW) pullets. Two egg-layer strains were used in the present study to determine how different genotypes are affected by infrared beak treatment. This work was approved by the University of Saskatchewan’s Animal Research Ethics Board (AUP 19940248) and adhered to the Canadian Council on Animal Care guidelines for humane animal use .n = 100 per strain) were obtained from a commercial hatchery. Prior to arrival at the research facility, 50 chicks per strain were infrared beak treated immediately post hatch (IR) by a trained operator. The remaining chicks were sham untreated controls (C), meaning that they were handled and placed into the infrared beak treatment equipment, but their beaks were not exposed to the infrared light. Infrared treatment settings for each strain are described in n = 44 per treatment) were housed in experimental cages (n = 44) from 0 to 21 days of age with 4 chicks per cage (625 cm2 per chick). Research staff were not blinded to which treatments the chicks belonged to. All pullets had ad libitum access to commercial chick starter and water. Feed was provided via chick feeders for the first 14 days and then in trough feeders for the remaining time. Water was provided through 360° nipple drinkers (2 nipples per cage), with supplemental waterers provided for the first 14 days. The photoperiod was 23L:1D (20 lux) for the first 7 days and then 8L:16D (15 lux) from day 8 onwards using incandescent light bulbs as the light source and included dawn and dusk periods of 15 minutes each. Heat was provided via hot water pipes running along the walls of the room. Room temperature started at 32 °C at 1 day and decreased by approximately 2 °C each week to reach a final room temperature of 25 °C at 21 days of age.Newly hatched LB and LW female chicks  ). Using the beak healing classifications described in p ≤ 0.05 and a trend was noted when 0.05 < p ≤ 0.10.The experiment was designed as a 2 × 2 factorial arrangement of beak treatment and strain, in a completely randomized design with 44 replicates per treatment (bird as replicate unit). Beak length data were analyzed using PROC MIXED  with Tukey’s range test to separate means. The non-parametric ordinal histological score data (infrared treated beaks only) were analyzed as a one-way analysis of variance of strain using PROC NPAR1WAY  and the Kruskal–Wallis test, with 88 replicates per strain. Differences were considered significant when Sloughing of the treated beak tissue was initiated at 10 days of age in approximately 2 percent of pullets and was complete by 20 days of age . For botp = 0.096). At 21 days of age, C pullets also had longer top beak lengths compared to IR pullets . The strains demonstrated differences in top beak length with LB pullets having longer top beaks than LW at 1 day of age, but shorter top beaks at 7 days of age . LB pullets had less overall beak growth compared to LW pullets over the 21 days . At 1 day post treatment, all treated beaks, regardless of strain, showed coagulative necrosis of the beak tissue below the treatment line . CellulaInfrared beak treatment had a quick impact on the beak tissue, as a trend appeared for IR pullets to have shorter top beak lengths compared to C pullets at 1 day of age. Similar findings have been reported by Henderson et al.  and StruOne of the advantages of infrared beak treatment is that it does not result in the immediate loss of the beak tip. The infrared beak treatment process worked as expected with treated birds having shorter beak lengths and less post-treatment beak growth once sloughing of the necrotic beak tip was initiated. Epithelial regeneration began prior to the initiation of beak sloughing, which reduces the chance that birds will have open wounds at the end of the beak once sloughing is complete. There was no evidence of neuroma formation or abnormal nerve growth, both of which are associated with chronic pain. Overall, the results of this study support the continued use of infrared beak treatment to help improve laying hen welfare and reduce cannibalism until effective and reliable alternatives to beak treatment are found.


Supplemental Digital Content is available in the text. The objective of this study was to test the ability to achieve, maintain, and subjectively benefit from extended high-frequency amplification in a real-world use scenario, with a device that restores audibility for frequencies up to 10 kHz.A total of 78 participants (149 ears) with mild to moderately-severe sensorineural hearing loss completed one of two studies conducted across eight clinical sites. Participants were fitted with a light-driven contact hearing aid (the Earlens system) that directly drives the tympanic membrane, allowing extended high-frequency output and amplification with minimal acoustic feedback. Cambridge Method for Loudness Equalization 2 - High Frequency (CAM2)-prescribed gains for experienced users were used for initial fitting, and adjustments were made when required according to participant preferences for loudness and comfort or when measures of functional gain (FG) indicated that more or less gain was needed. Participants wore the devices for an extended period. Prescribed versus adjusted output and gain, frequency-specific FG, and self-perceived benefit assessed with the Abbreviated Profile of Hearing Aid Benefit, and a custom questionnaire were documented. Self-perceived benefit results were compared with those for unaided listening and to ratings with participants’ own acoustic hearing aids.The prescribed low-level insertion gain from 6 to 10 kHz averaged 53 dB across all ears, with a range from 26 to 86 dB. After adjustment, the gain from 6 to 10 kHz decreased to an average of 45 dB with a range from 16 to 86 dB. Measured FG averaged 39 dB from 6 to 10 kHz with a range from 11 to 62 dB. Abbreviated Profile of Hearing Aid Benefit results revealed a significant improvement in communication relative to unaided listening, averaging 28 to 32 percentage points for the background noise, reverberation, and ease of communication subscales. Relative to participants’ own hearing aids, the subscales ease of communication and aversiveness showed small but significant improvements for Earlens ranging from 6 to 7 percentage points. For the custom satisfaction questionnaire, most participants rated the Earlens system as better than their own hearing aids in most situations.Participants used and reported subjective benefit from the Earlens system. Most participants preferred slightly less gain at 6 to 10 kHz than prescribed for experienced users by CAM2, preferring similar gains to those prescribed for inexperienced users, but gains over the extended high frequencies were high relative to those that are currently available with acoustic hearing aids. Most conventional hearing aids provide useful gain for frequencies up to 4 to 5 kHz . There hStudies on the usefulness of amplification at HFs have differed in the definition of HF, the types of participants, the specific methods used, and the outcome measures used, leading to mixed conclusions about the potential usefulness and profile of users who may benefit from extended HF amplification , 2011. UThe Earlens system allows exploration of the potential benefits of extended HF hearing for individuals with hearing loss in real-life settings. This article describes the results of initial evaluations of the fitting and efficacy of the Earlens system when using the CAM2 prescription, based on real-world use across eight sites by a large cohort of participants. Data were obtained during two prior clinical trials where the main goals were the assessment of safety and efficacy. However, additional data were collected and are used in new analyses reported here to explore the typical gains and output levels that were prescribed and achieved over the extended HFs and to report the adjustments from computed targets that were required based on participant reports after real-world field experience. Gains and output levels prescribed by CAM2 for the initial fit are compared with the corresponding levels after adjustment to align with functional gain (FG) expectations and individual participant preferences, and changes are discussed in terms of their clinical implications. In addition, self-perceived benefit outcomes and satisfaction ratings for the adjusted fittings are used to examine the relationship between subjective outcomes, programmed gain settings, and audiometric characteristics. This information is a first step towards further exploration of the possible benefits of providing extended HF audibility to hearing aid users.Data for this study were collected during the course of two nonblinded clinical trials of the Earlens system that were conducted under a Food and Drug Administration (FDA) investigational device exemption. A complete description of the clinical trial and outcomes of the first study, which was intended to demonstrate the safety and effectiveness of the Earlens system to support FDA clearance, can be found in This article presents additional data collected during the trials and analyzes relationships between the additional data and prior efficacy data. Data are presented for unaided listening (collected before the devices were fitted), aided results at first fit or 1 week post-first fit, and aided results collected at the nominal end point of each study: at 90 days (study 2) or 120 days (study 1). FG data from study 1 have previously been reported as a secondary endpoint from testing at the 30-day interval . FG dataParticipants were recruited from various sites using institutional review board–approved materials, including from nearby private audiology practices, via existing patients of Earlens Corp, and by word-of-mouth. They were required to meet the medical and anatomical criteria for wearing the Earlens system. Those enrolled were between 33 and 84 years old, with an average of 68 years. All participants were fluent speakers of American English, and all but four were native speakers of American English. Participants had mild to severe sensorineural hearing loss in one or both ears see Fig. , suprathhttp://links.lww.com/EANDH/A476, which details the questions and response scales for each study that are discussed in the current article). Responses were made on an ordinal 5- or 6-point Likert scale spanning a range from negative to positive, with each point labeled with a descriptor. The satisfaction questionnaire was revised between the two studies; individual questions were updated, and the response scale was changed to allow for a neutral response at the center of the scale.Audiological evaluations were conducted using the study site’s clinical equipment . Equipment models varied by site, but all had been calibrated within the past year, including frequency-specific stimuli from 9 to 10 kHz for audiometers with testing capabilities at those frequencies. The audiometer was used to present pure-tone signals for air- and bone conduction threshold measurements, recorded NU-6 word lists , and warble tones for sound field threshold measurements. Self-perceived benefit was assessed with the Abbreviated Profile of Hearing Aid Benefit (APHAB) questionnaire , a validSubjective comparisons of Earlens to participant’s own air conduction hearings aids are reported for the APHAB and some items on the satisfaction questionnaire. Participants’ own hearing aids were evaluated in the “as is” condition as these studies were not intended primarily as comparison studies but rather to establish the safety and efficacy of the Earlens system.As illustrated in Figure The processor includes a 20-channel compression system, with an attack time of 50 ms and a release time of 1000 ms in each channel. A constant compression ratio is applied for levels between the compression threshold (CT) and the level at which compression limiting occurs, which is referred to as the maximum equivalent sound pressure output (MPO), similar to the MPO for acoustic hearing aids. Expansion is applied for levels more than about 6 dB below the CT. Fast-acting compression limiting is applied for output levels above the output compression limiting threshold in each channel. A feedback cancellation system can be activated to achieve additional stable gain if required. The processor is programmed using Hi-Pro 2 interface hardware and proprietary software that is similar in function to that for acoustic hearing aids, but with two additional features: a light calibration procedure and FG mode, described later.Participants were enrolled after agreeing to be included in the study via standard informed consent procedures. The following measures were obtained using standard clinical procedures for each ear to determine whether the candidacy criteria were met: air conduction and bone conduction audiometric thresholds (using 2-dB steps for study 1 and 5-dB steps for study 2); word recognition scores for recorded NU-6 word lists at the presentation level giving the highest score, PBmax (50-word list for study 1 and 25-word list for study 2); and tympanometry. Baseline unaided sound field thresholds were measured for each ear with the nontest ear plugged or masked as needed,f. The previously measured audiometric threshold at each frequency in dB HL was converted to the equivalent in dB SPL, denoted Tf. In this way, it was known that a light intensity of If would give a sound level equivalent to Tf. This procedure is conceptually equivalent to calibration of an air conduction hearing aid using a probe microphone in the ear canal although the uncertainty of the calibration at HFs is much less for the light-calibration procedure than for probe-microphone measurements due to the absence of ear-canal standing waves that affect probe-microphone measurements. The light-calibration procedure combined with the maximum possible value of intensity of the light was used to determine the maximum possible equivalent sound pressure output (MPPO) of the system on each ear at each frequency. This is different from the MPO, mentioned earlier, as the MPO was adjustable for each ear in the fitting software; the MPO was always equal to or below the MPPO. After the system was calibrated in this way, participants were fitted with the CAM2 prescription for frequencies up to 10 kHz, using the Earlens fitting software. All participants were initially fitted with the “full” gains recommended by CAM2 for experienced users of hearing aids. Gains were then either manually adjusted or automatic acclimatization (when available) was enabled based on informal participant feedback. Recent versions of the CAM2 software incorporate reduced gains for inexperienced users of hearing aids , but the version used for the studies reported here did not incorporate this option.After placement of the tympanic lens, a calibration procedure was performed for each ear to measure the effective output in response to a calibrated light signal as a function of frequency. For this purpose, sinusoidal signals were synthesized and delivered as pulse-density modulated light. The intensity of the light required for the signal to be detected was measured for each frequency. We denote the light intensity at threshold for a given frequency as IThe fittings were adjusted to provide comfort and to reduce acoustic feedback when necessary. In study 1, only minimal adjustments were made for feedback before measurement of FG at the first fit visit , but more substantial adjustments were sometimes made after measurement of FG at the first fit visit, especially when the FG appeared to be markedly lower or higher than expected from the programmed gains (these adjusted program settings are termed “First Fit Adjusted”). In study 2, no First Fit Initial measurements were made as the study was designed to more closely mimic normal commercial flow, and therefore adjustments were permitted before the measurement of FG (these measurements are termed “First Fit Adjusted”). In study 2, acclimatization became available in the fitting software mid-study, and it was activated for one third of the participants. When activated, the programmed gains were initially set below the target values, either for all frequencies or only for the extended HFs (6 to 10 kHz). The gains were then gradually increased towards the target values over a period of between 1 and 6 weeks, depending on the autoacclimatization settings chosen by the audiologist.Aided sound field thresholds were measured for each ear as described for the unaided condition, by placing the device in a “functional gain mode” which programs the device to be linear (no compression) and sets the insertion gain (IG) in each channel to the IG at the Compression Threshold (IGCT) for the current fitting. The IGCT represents the maximum IG that the device would apply at each frequency, and it depends on both the hearing level of the individual and the spectrum of “soft” (50 dB SPL) speech. FG at each frequency was calculated as the difference between the baseline unaided sound field threshold and the aided sound field threshold. Measures of FG in the sound field were compared with the prescribed and programmed IGs at multiple time points to “verify” the fitting as real ear measures are not possible with this nonacoustic device.The APHAB questionnaire for the aided Earlens condition was administered at 90 or 120 days post-fitting. The Earlens-designed Satisfaction Questionnaire was also administered at 90 or 120 days post-fitting. The programming software fitting files were saved at each time point so that prescribed and adjusted (current fit) output and gain data could be extracted and analyzed.Not all subjects completed all measurements at all test intervals. Five participants who did not complete each study were removed from the current analysis. In addition, among those who completed the study, there were a few instances of incomplete data sets, usually due to missing aided sound field data at first fit or at the endpoint of the study, or occasionally a missing fitting file. These instances occurred across multiple test sites. Thus, data from six additional ears were removed from the output and gain analyses of study 1, and 11 additional ears were removed from the same analyses for study 2. Therefore, the current analysis includes 42 participants (80 ears) for study 1 and 36 participants (69 ears) for study 2 with complete study data. In the case of APHAB and satisfaction data, which are participant-based instead of ear-based, the data of these participants were also removed, including those fit bilaterally but with complete data in only one ear. In addition, subjects who wore their own hearing aids before the study were also tested with those aids, leading to different numbers of subjects in comparisons of Earlens to unaided and Earlens to own hearing aid. All comparisons that were analyzed statistically used paired-comparison methods based only on participants with data for both variables involved in the comparison, and only those participants’ data are plotted.The data extracted from the fitting software include effective output level in dB SPL for “soft” (50 dB SPL), “moderate” (65 dB SPL), and “loud” (80 dB SPL) input levels of speech. The effective output level at each center frequency was calculated by integrating the speech spectrum power across one equivalent rectangular bandwidth (ERB) of the auditory filter centered at that frequency. The value of the ERB for each participant and center frequency was estimated taking into account the hearing loss of the participant at that center frequency, as described by Figure The right panel of Figure Figure For study 2, the 90-day time point was the nominal end of the study. Despite automatic acclimatization, which was complete before 90 days, the 90-day output values were only slightly higher than the First Fit Adjusted values for the extended HFs .The measures of FG at each test frequency should be closely related to the values of IGCT programmed in the fitting software. However, the two will not be numerically identical. The measures of FG were obtained in sound-treated rooms using a loudspeaker with 0° azimuth and elevation, thus approximating free-field listening conditions. However, the IGs prescribed by CAM2, including the maximum insertions gains, IGCT, are based on diffuse-field listening . The twoFigure The First Fit Adjusted gain values for study 2 are shown in Figure The current version of the CAM2 software includes a modified prescription for inexperienced hearing aid users . Although most of the participants in the current study were experienced hearing aid users, they had not previously experienced amplification over the extended HF range (6 to 10 kHz), so, at least for that frequency range, the gains recommended for inexperienced users might be more appropriate. To assess this, we compared the CAM2 inexperienced-user IGCT values to the current fit IGCT values obtained after the adjustment at first fit, to see if the adjustments that the audiologists made led to IGs similar to those prescribed by CAM2 for inexperienced users. The outcomes for study 1 are shown in Figure p value of 0.05. A two-factor repeated-measures analysis of variance was performed with the factors: listening condition  and subscale . There was a significant main effect for both listening condition  and subscale , and the listening condition × subscale interaction . Communication problems for the background noise, reverberation, and ease of communication subscales decreased by 28 to 32 percentage points when fitted with Earlens relative to the unaided condition. Sidak multiple comparisons test revealed that these differences were significant  for each comparison. For aversiveness, there was a significant increase of 7.5 percentage points for Earlens relative to unaided . The finding for aversiveness is not unusual as many studies of hearing aids have shown an increase in aversiveness relative to unaided listening  and subscale . There were significant main effects of listening condition , subscale , and the interaction . Tukey’s multiple comparisons tests revealed that relative to the unaided condition, there was a significant decrease in communication problems on the background noise, reverberation, and ease of communications subscales for both Earlens and own HA . Also, aversiveness increased for both own HA and Earlens relative to unaided (p < 0.001 for each), by 16 percentage points for own HA and by 9 percentage points for Earlens. The difference between own hearing aids and Earlens was not significant for background noise , or reverberation , but was significant for ease of communication  with an average decrease of problems of 6.4 percentage points for Earlens. There was also a difference for aversiveness , with Earlens showing an average decrease in problems of 7.3 percentage points relative to own HA.The APHAB questionnaire was administered at baseline before fitting for unaided listening and to assess experiences with the participants’ own hearing aid(s), which applied to most participants. Experience in listening with the Earlens system was evaluated at the end point of each study. APHAB data were pooled between the two studies for this analysis to give increased statistical power. The upper panel of Figure istening . The lowon [F1, 6 = 187.2,http://links.lww.com/EANDH/A476: Relevant Excerpts of the Earlens Patient Satisfaction Questionnaires. These include questions relating to satisfaction  and perceived benefit relative to unaided listening and listening with own hearing aids across a variety of situations. Statistical analyses were not performed on these data due to lack of power, particularly for study 2, and because there was no straightforward way of combining data across studies due to the different questions and response scales. In addition, the questionnaire was not validated.The specific questions for which the responses were analyzed are contained in the Supplemental Digital Content 1, Median satisfaction ratings and upper and lower quartiles for study 1 are shown in Figure x axis. The upper panel is for comparison to unaided listening while the lower panel is for comparison to the participants’ own hearing aids. For both studies, the median ratings for all listening situations were clearly above the neutral rating of “No change” or “About the same,” relative to both unaided listening and listening with their own hearing aids. Ratings compared with unaided hearing were generally better than those compared with own hearing aids, as expected. However, the results indicate that the Earlens system was generally rated as better than the participants’ own hearing aid(s). For study 1, the lower quartile was above “No change” for most listening situations while for study 2, the lower quartile of ratings reached the “About the same” rating for most categories.Participants were also asked how the Earlens compared with listening unaided or listening with the hearing aid(s) that they used before the study. The median and upper and lower quartiles of the responses are shown in Figure In fitting any hearing aid for a person with sensorineural hearing loss, there is often a compromise between fully matching prescriptive targets to optimize the audibility of speech sounds and meeting the user’s preferences for loudness and sound quality. In addition, most people with HF hearing loss lack experience with amplification over the extended HF region. While the Earlens system is able to provide substantial HF and extended HF gain, it was not initially clear whether users would like it and perceive benefit from it and how perceived benefit might vary with the type and amount of hearing loss. The answers to these questions can help determine best-practice gain settings for optimizing satisfaction. In addition, although the Earlens fitting range is quite wide, it is possible that individuals with certain audiometric characteristics have greater subjective benefit and preference for Earlens. This led us to conduct a correlational analysis to assess whether self-perceived benefit and satisfaction were related to measures, such as audiometric parameters of the underlying hearing loss (pure-tone average and slope), measured FG, or adjustments made to the fitting over or under prescriptive targets. There were no predetermined theories about which hearing profile might benefit most or least or whether fitting at or under targets would have a positive or negative effect on subjective ratings. The correlation was determined between each of several objective summary measures and each of several subjective summary measures. The objective measures were as follows: pure-tone average (PTA) for LF (0.125 to 1 kHz), HF 2 to 10 kHz), and broadband (broad = 0.125 to 10 Hz); slope for LF, HF, and broad; FG for LF, HF, and broad; and mismatch (the difference in output between the CAM2 prescription and the current fitting for speech with an input level of 65 dB SPL) LF, HF, broad, speech frequencies (Sp Freq = 0.5 to 4 kHz), and extended HF (6 to 10 kHz). The subjective measures were as follows: APHAB global benefit for Earlens (relative to unaided listening or listening with the participants’ own hearing aid(s), HA) averaged across the background noise, reverberation, and ease of communication subscales; and average satisfaction, perceived benefit re-unaided listening, perceived benefit re-own hearing aid(s), and preference re-own hearing aids. The last four measures were based on averages across responses from applicable questions on the Earlens Patient Satisfaction Questionnaire.p < 0.05 with a correlation coefficient down to ≈0.25. Significance levels were not corrected for multiple testing as we considered this only a preliminary exploration. Even without correction, most of the correlations were not statistically significant. Only slope LF was weakly correlated with APHAB benefit re-unaided, such that as the slope increased, benefit re-unaided increased. Correlations between the same objective measures and average satisfaction ratings, perceived benefit, and preference re-own hearing aid were calculated separately for studies 1 and 2 as these data could not be easily pooled. The power was only sufficient to obtain significance for correlations above 0.32 to 0.35 due to smaller sample sizes . None of the objective measures were significantly correlated with the subjective outcomes. The fact that all correlations were small indicates that the objective measures did not account for much of the variability in subjective responses. Different results might have been obtained with a larger sample size.The results are shown in Table The Earlens system was able to provide the gains and output levels prescribed by the CAM2 fitting method for a wide range of hearing losses and over the whole frequency range up to 10 kHz. Peak output levels averaged 123 to 124 dB SPL, similar to those for the temporal bone data of The APHAB results suggested that problems with aversiveness were significantly less for Earlens than for the participants’ own hearing aids. This happened despite the fact that the former provided much more gain than the latter over the extended HFs. Evidently, the restoration of audibility for the extended HFs did not come at the cost of an overall increase in aversion.Although the majority of the participants were previous hearing aid users, the final gains for the extended HF range were closer to the new-user settings than to the experienced-user settings from the CAM2 fitting algorithm. Sometimes, the HF gains were slightly reduced to avoid acoustic feedback, but the main reasons for the adjustments were participant reports of sharp sound quality, harsh onsets, sounds being too loud or uncomfortable, and distortion of /s/ and other HF sounds. These findings may indicate a need to modify the CAM2 fitting method to use somewhat reduced gains over the extended HF range. It may be the case that the gains recommended for new users are actually appropriate even for experienced users in that frequency region. Alternatively, these findings may reflect lack of familiarity with extended HF audibility as air conduction hearing aids, even if fitted to targets, usually do not provide audibility in that frequency region. Activation of acclimatization in the hearing aid programming may increase the likelihood that participants prefer the CAM2 targets for experienced users. In the current study, the subset of participants who had acclimatization activated was relatively small, and their fully-acclimatized settings at the 90-day visit were still ≈10 dB below the prescribed output at 8 to 10 kHz. A different acclimatization algorithm, for example, with a longer time course or smaller step size, may be more effective.As described earlier, the initial fitting of the Earlens system involves a light-calibration procedure that is conceptually similar to real ear measurement for conventional air conduction hearing aids. However, unlike the latter, the light-calibration procedure for the Earlens system is obligatory; the fitting cannot proceed until the light-calibration has been completed. Thus, the person doing the fitting cannot save time by skipping the procedure. Furthermore, once the light-calibration procedure has been completed, the target gains can be implemented very accurately, probably more accurately than with conventional air conduction devices, for which the achieved gains often differ markedly from the target gains .Although acoustic feedback can occur with the Earlens system, it is much less of a problem than for conventional air conduction hearing aids because sound radiation from the ear canal is small when the TM is driven directly . The defIndividual differences in subjective ratings of the benefit of the Earlens system relative to unaided listening or to their own hearing aids as determined using the APHAB questionnaire see Fig.  or in thA majority of the participants in the two studies had audiometric thresholds that did not exceed 65 dB HL for frequencies up to 4 kHz see Fig. . It is uThe FGs measured at the start of each study corresponded reasonably well with the programmed IGs for soft speech for frequencies up to about 4 kHz, as should be the case if the system is calibrated accurately  of the National Institutes of Health. Portions of the data in this article were presented at the International Hearing Aid Research Conference (IHCON), Tahoe City, CA, August 10–14, 2016. Results from three Earlens Patient Satisfaction questions from study 1 were presented in a different format in


The chinchilla animal model for noise-induced hearing loss has an extensive history spanning more than 50 years. Many behavioral, anatomical, and physiological characteristics of the chinchilla make it a valuable animal model for hearing science. These include similarities with human hearing frequency and intensity sensitivity, the ability to be trained behaviorally with acoustic stimuli relevant to human hearing, a docile nature that allows many physiological measures to be made in an awake state, physiological robustness that allows for data to be collected from all levels of the auditory system, and the ability to model various types of conductive and sensorineural hearing losses that mimic pathologies observed in humans. Given these attributes, chinchillas have been used repeatedly to study anatomical, physiological, and behavioral effects of continuous and impulse noise exposures that produce either temporary or permanent threshold shifts. Based on the mechanistic insights from noise-exposure studies, chinchillas have also been used in pre-clinical drug studies for the prevention and rescue of noise-induced hearing loss. This review paper highlights the role of the chinchilla model in hearing science, its important contributions, and its advantages and limitations. I.Chinchilla lanigera), has been used as an animal model for studying auditory function and the effects of hearing loss for many decades, with references dating back to the early 1960s. Chinchillas are indigenous to the Andes Mountains of South America, where they live in herds. The name chinchilla means “little chinchas,” which is a reference to the Chincha people who lived in the same mountain range. Chinchillas were first domesticated when they were brought to the United States in the early 1920s by Mathias F. Chapman, a US-American mining engineer working in Chile. At that time, chinchillas were protected by the Peruvian, Bolivian, Chilean, and Argentine governments, so Chapman was granted special permission to bring back 11 chinchillas  to the United States to begin a breeding population. Today, there are hundreds of chinchilla ranches in the US, some of which supply chinchillas for auditory research whereas others raise chinchillas for the furrier industry.The chinchilla, or more specifically the long-tailed chinchilla  enacted in 1966; provisions to this act are enforced by the Department of Agriculture (USDA). Thus, research using chinchillas is strictly regulated and must be scientifically justifiable over lower species. A number of anatomical, physiological, and behavioral advantages do exist that justify the use of chinchillas for auditory research and have made it an extremely valuable animal model for hearing science. Primary among these advantages are the similarity of their hearing frequency range and sensitivity to humans and their genetic heterogeneity compared to other rodent models. Additionally, the chinchilla's docile nature and long lifespan  make it suitable for short- or long-term studies. The ability to measure and relate a breadth of anatomical, behavioral, and physiological effects of various noise exposures within the same species has led to widespread use of the chinchilla as a model for noise-induced hearing loss (NIHL). The chinchilla's history as an animal model of acoustic injury has also led to a number of studies exploring pharmacological rescue and prevention using antioxidants and other biologically active compounds and drugs aimed at preventing or limiting the effects of noise trauma.The overall aims of this paper are to (1) highlight the historical relevance of the chinchilla model in hearing-science research, including its strengths and limitations, (2) review the breadth of key findings related to noise exposure that demonstrate the usefulness of the chinchilla animal model, and (3) highlight some of the subsequent chinchilla studies evaluating the ability of pharmacological agents to protect against NIHL.II.A.An important advantage that chinchillas have over many other rodent models is that their hearing sensitivity and frequency range significantly overlaps with humans Fig. , with thAnatomical similarities with humans include a similar number of cochlear turns , a similar variation in cochlear length , and a wide tympanic membrane  permits behavioral performance to be assessed using operant conditioning for simple and complex stimuli, as well as for physiological data collection from awake animals . Likewise, anatomical and physiological characteristics in chinchillas have allowed for robust data collection along the entire auditory system using both invasive and non-invasive techniques. Thus, the widespread use of chinchillas in NIHL studies Sec.  has been1.Chinchillas are relatively easy to train and have been used extensively in behavioral studies of sound detection and discrimination. These studies include experiments on pitch/intensity detection and discrimination, signal detection in noise, temporal changes in acoustic cues, and sound localization. Their long lifespan relative to other rodents, combined with their intelligence, make long-term behavioral experiments feasible . Chinchillas can be used to measure both operant as well as reflexive responses to acoustic stimuli. Operant conditioning is a preferred method because once trained, the animals are reliable attending observers under stimulus control. This attention permits implementing the types of complex tasks that are necessary to test both threshold and suprathreshold hearing assessments pre- and post-intervention. Operant conditioning studies with chinchillas have used negative-reinforcement  as well as positive-reinforcement approaches . However, there are no peer-reviewed studies using reflexive measures in the chinchilla in response to acoustic stimuli . It is possible that the paucity of startle studies in chinchillas is due to long release times from the startled state, or the presence of only subtle indicators of a startle response similar to those observed in the guinea-pig post-auricular response  testing, a robust measure in chinchillas related to the non-linear gain provided by outer hair cells (OHCs). In chinchillas, a linear relation between DPOAE amplitude and percentage of OHC loss was established even when OHC lesions were relatively broad . Auditor4.The chinchilla's docile nature and durability to chronic surgeries provide further significant advantages that allow for a wide range of physiological measurements to be made from restrained awake animals . For exaElectrophysiological measures from the round window during long-duration noise exposure have also been made to evaluate the protective nature of the middle-ear muscle reflex  neurophysiological single-neuron experiments e.g., . Due to An enormous dataset characterizing anesthetized chinchilla AN-fiber tuning properties  has been studied in chinchillas in terms of the head-related transfer functions . SpecifiSupplementing these higher-order findings, data from auditory-cortex neurons in chinchillas were used to explore which aspects of sensitivity to FM sweeps, a fundamental building block of speech, are experience-dependent in development . The abiFurthermore, important and significant effects of NIHL have been shown to be dependent not only on the recording site, but also on the acoustic stimulus used. For instance, unlike broadband-noise stimuli, narrowband stimuli can fail to capture the distorted tonotopic organization often found following noise exposure , underlying health problems can create unexpected complications and potential attrition that may occur more frequently than with other common research rodents. Furthermore, due to their less frequent use in research, veterinarians and their staff are generally less experienced with chinchilla medical care than other rodent species. This limitation can result in difficulty with early detection of illness and prescribing appropriate treatment. Under many circumstances, chinchillas require care by an exotic animal veterinarian. It should be noted, however, that despite these potential limitations, many long-term studies of behavior and noise exposure have been carried out successfully in chinchillas, as discussed throughout this review.Another important consideration is that chinchillas must be housed in a manner that differs from most laboratory rodents. For example, because chinchillas have 60–90 hairs per follicle, they are at higher risk for overheating and must be kept at cool temperatures and low humidity levels. Ideally, chinchillas should be kept in housing conditions that resemble their native mountain habitat . Temperatures above 75 °F can become fatal. Compared to other rodents used in research, such as mice and rats, chinchillas also require more elaborate diets that are rich in fiber and protein in order to maintain healthy gastrointestinal systems. Chinchillas also routinely require dust baths to absorb oils and moisture buildup in their fur. These baths mimic dust available in their natural habitat that helps keep their fur clean and healthy.It is also important to note that the chinchilla's 112-day gestation period is substantially longer than the 21-day gestation period for rats and mice. Furthermore, one gestational period only yields 1–2 kits. Weaning a chinchilla kit takes about six weeks, compared to rats and mice that only require three weeks. These limitations make it much more difficult to breed and raise chinchillas in research studies for which well-controlled acoustic environments are desired from birth. Chinchillas are also born with a fully developed auditory system, which may or may not be advantageous to the research of interest and could significantly limit developmental studies of the auditory system.Anatomically, chinchillas have a thin bulla and skull. Though this allows easy access to the cochlea and other structures, it can also create a challenge for mounting implanted chronic electrodes and electrode arrays. Differences in outer and middle ear acoustics may contribute to differences in noise susceptibility between chinchillas and other species . ChinchiFinally, when considering the use of chinchillas for research purposes, it is crucial to remember their USDA status as a protected species. Per the AWA, the use of chinchillas must be scientifically justifiable over lower species, which can make approval from Institutional Animal Care and Use Committees more difficult than for other rodent species.D.In addition to studies of NIHL  can be used to produce selective IHC damage uniformly along the cochlea [e.g., Fig. The carboplatin model provides a complement to the selective OHC-loss models that antibiotics produce in many species, including chinchillas . For exaBeyond the use of ototoxic drugs to create selective hair-cell models to dissect the effects of NIHL, chinchillas have also provided a useful animal model for testing specific drugs for ototoxicity. Alternative antibiotics have been tested for ototoxicity in chinchillas when the widespread use of non-ototoxic antibiotics has led to the development of antimicrobial resistance  to determine occupational-exposure guidelines for noise  will higA.Many anatomical studies in the chinchilla have evaluated the degeneration of sensory hair cells in the inner ear as a function of noise trauma e.g., . When exOther studies using less-intense noise exposures on chinchillas highlight an additional form of micro-structural cochlear damage that can occur following noise exposure. Recent immunohistochemical studies in mice have demonstrated that moderate-noise-exposures that produce a TTS can produce permanent cochlear synaptopathy, which is a degeneration of afferent synapses that may contribute to a latent form of hearing loss characterized by difficulties listening in noise despite an otherwise normal audiogram . The samB.Noise-exposure studies in chinchillas have demonstrated that high-level exposures that cause hair-cell loss also decrease behaviorally derived estimates of hearing sensitivity e.g., . The oveIn addition to elevated thresholds, hearing loss also produces other functional deficits. One of the most common is the loss of tuning, i.e., poorer frequency selectivity. Reduced frequency selectivity is thought to underlie the ability to hear in competing background noise . This maThe effects of noise trauma on tuning characteristics (measured using simultaneous tone-on-tone masking) have been studied in chinchillas with evoked potentials assessed along the auditory pathway , results showed that non-invasive methods could predict both the frequency selectivity and threshold responses of afferent AN fibers   was proposed by the US Army Aeromedical Research Laboratory using data from human volunteers in a variety of conditions and previous models (Ensuing studies sought to characterize the degree of TTS and PTS that occurs from impulse noise by looking at asymptotic threshold shift (ATS) during long-lasting  noise exposures  for 1, 10, or 20 days. Each of the conditioned groups showed significantly less PTS than the non-conditioned groups, with no significant differences in PTS (or hair-cell damage) between the 1-, 10-, and 20-day groups  showed less PTS and hair-cell loss Fig.  after a In summary, chinchilla conditioning experiments have demonstrated the following: (1) the ear can indeed be toughened by lower-level pre-exposures; (2) these effects can be long-lasting; and (3) these effects convey protection against other types of noise such as impulses. It is important to note that the precise mechanisms underlying these “conditioning” effects are not well understood. However, conditioning studies have provided initial insight into underlying mechanisms that has led to investigations into potential strategies for prevention and rescue.In chinchillas and other species, noise exposures result in increased levels of reactive oxygen species (ROS) as a metabolic byproduct of high levels of cochlear activity during noise. The high levels of ROS have been shown to potentiate damage to the inner ear; however, ROS activity can be mediated by endogenous antioxidant activity. When antioxidant enzyme levels were compared between conditioned animals (with less hearing loss) and non-conditioned animals (with more hearing loss), conditioning was shown to enhance the endogenous antioxidant glutathione (GSH), in addition to catalase .Furthermore, ear-toughening experiments are particularly interesting given the current state of science as it pertains to synaptopathic hearing loss . In otheH.In the section above, the effects of previous noise exposure were shown to reduce subsequent NIHL. However, NIHL can also be augmented. Industrial solvents such as toluene and styrene have been repeatedly shown to potentiate NIHL in animal models , though female representation has slowly increased over time. As an example, as of April 2018, women represent just 16% of the US Military, a number that shows significant increase from the 2% reported in 1973 . The oveRecent large-scale prospective cohort studies have looked at the differences in average hearing loss of adults across age and sex and compared the results to previous studies that had done the same . AdditioOverall, chinchillas have been used sparingly as an animal model in this context. One such study exposed a group of male and female chinchillas to an impulse noise reaching approximately 150 dB SPL, then re-assessed their auditory sensitivity at various intervals post exposure to determine TTS and PTS levels  differences that could make males more susceptible to NIHL than females. These differences have been seen in anatomical damage as well as functional hearing ability, but an understanding of why these differences might exist is not yet clear. The results found in the studies mentioned parallel the nominal human research addressing the same topic and highlights the chinchilla as an acceptable animal model to further investigate sex differences in NIHL.K.Evidence continues to accumulate that threshold-shift effects of hearing loss only partially account for the perceptual effects that people with hearing loss face in daily life, particularly in noisy environments. Thus, as NIHL and prevention/rescue studies advance, it will become all the more important to consider suprathreshold effects of noise exposure on behavior and neural coding of perceptually relevant sounds, in addition to anatomical and physiological effects. Inasmuch as behavioral techniques have been largely replaced with objective physiological measures for threshold effects, for experiments that use complex auditory stimuli , operant behavioral techniques may be more appropriate where objective physiological measures may not adequately assess study outcomes.Chinchillas have long been used successfully to evaluate normal-hearing behavioral acuity using complex stimuli  or can be impulsive and unexpected . Occupational hearing conservation programs exist to monitor hearing and to provide hearing protection devices (HPDs) to prevent long-term hearing loss. However, in situations such as military operations, these HPDs can be either impractical or ineffective for the types of acute acoustic trauma (AAT) experienced in the field. Current military protocols or treatments for AAT are largely impractical  and/or are not completely effective or are associated with serious side effects . Thus, pharmacological strategies are of great interest for protection and rescue from NIHL. A number of pharmacological agents have been motivated by ear-conditioning experiments  neural degeneration at the level of the cochlear nucleus has been reduced as a result of administering a combination of the antioxidants NAC and ALCAR  mediated apoptosis, reduced PTS to 155 dB SPL impulse noise from 16–25 dB to 6–17 dB after a single administration at 1 to 4 h post-exposure  history as a valuable animal model for hearing science and NIHL. They are especially useful when comparisons of anatomical, physiological, and behavioral effects in the same species are important. Chinchillas are relatively easy to train, their docile nature permits awake physiological measures, their anatomy allows for direct physiological measurements along the entire auditory pathway, and their lifespan allows for long-term studies. Thus, the chinchilla model is flexible in accommodating both short- or long-term noise exposures as well as for studies employing continuous, impulse, or impact noise. Furthermore, potential pharmacological treatments can be delivered locally through a relatively accessible and thin round-window membrane, via intraperitoneal or intravenous injection, or orally. As reviewed here, these advantages of the chinchilla model have been and will continue to be effectively leveraged for research on the basic science of NIHL, the perceptual consequences of NIHL, and for evaluating potential therapeutic treatments to reduce or abolish NIHL.


Currently, l-ornithine production is focused on microorganism fermentation using Escherichia coli or Corynebacterium glutamicum. In C. glutamicum, development of high l-ornithine producing C. glutamicum was achieved by deletion of argF, but was accompanied by growth deficiency and arginine auxotrophy. l-Arginine has been routinely added to solve this problem; however, this increases production cost and causes feedback inhibition of N-acetyl-l-glutamate kinase activity. To avoid the drawbacks of growth disturbance due to disruption of ArgF, strategies were adopted to attenuate its expression. Firstly, ribosome binding site substitution and start codon replacement were introduced to construct recombinant C. glutamiucm strains, which resulted in an undesirable l-ornithine production titer. Then, we inserted a terminator (rrnB) between argD and argF, which significantly improved l-ornithine production and relieved growth disturbance. Transcription analysis confirmed that a terminator can be used to downregulate expression of argF and simultaneously improve the transcriptional level of genes in front of argF. Using disparate terminators to attenuate expression of argF, an optimal strain (CO-9) with a T4 terminator produced 6.1 g/L of l-ornithine, which is 42.8% higher than that produced by strain CO-1, and is 11.2-fold higher than that of the parent CO strain. Insertion of terminators with gradient termination intensity can be a stable and powerful method to exert precise control of the expression level of argF in the development of l-ornithine producing strains, with potential applications in metabolic engineering and synthetic biology.The online version of this article (10.1186/s13568-018-0557-8) contains supplementary material, which is available to authorized users. For l-ornithine production in C. glutamicum, a seed culture was prepared by inoculating cells into the seed medium  and allowing them to grow for 11 h. Then, the seed culture was inoculated into 25 mL of the fermentation medium and the initial OD600 was adjusted to 1. Each liter of the fermentation medium consisted of 100 g glucose, 20 g corn steep liquor, 50 g (NH4)2SO4, 2.5 g MgSO4·7H2O, 1 g KH2PO4, 0.5 g K2HPO4, 0.5 g Na2HPO4, 0.02 g FeSO4·7H2O, 0.02 g MnSO4·4H2O, and 10 g CaCO3  and inserted among the homologous arms by overlap PCR. The sequences of the synthetic RBSs are listed in Additional file argF, terminators derived from a previous study , and the RNA concentration was determined by a microplate reader . The cDNA was synthesized using the PrimeScript RT Reagent Kit with gDNA Eraser  and a Touch Real-Time PCR System , using the SYBR Premix Ex TaqTM II  on a Bio-Rad CFX96. cDNA (100 ng) was used as template. The PCR conditions were: 94 °C for 30 min, then 45 cycles at 94 °C for 5 s and 60 °C for 30 min. The data were normalized as per the 16S rRNA expression. The primers for RT-PCR are presented in the Additional file RT-PCR assays were conducted as described in our previous study  using an spectrophotometer, after dilution of the culture with 0.125 mol/L HCl to dissolve CaCO3 , plays a critical role in the biodegradation of l-ornithine. To accumulate l-ornithine, argF was deleted in strain CO, resulting in strain CO-1. Then, strain CO-1 was cultivated in a shaking flask, and the fermentation data revealed that 4.27 g/L of l-ornithine was detected in the broth after 48 h of incubation. However, the cell growth of CO-1 was 22% lower than that of the parent CO strain , and the start codon ATG was replaced with GTG, generating strains CO-2, CO-3, CO-4, and CO-5. As shown in Fig. l-ornithine production by these strains showed obvious improvement, compared to the CO strain, but the highest titer produced by strain CO-4 was only 1 g/L, which was three-fold lower than that produced by CO-1 (4.27 g/L). It was concluded that attenuating the expression of argF through RBS optimization and start codon replacement was undesirable for l-ornithine accumulation.Based on the results of the argCJBDFR operon, was disrupted previously, which led to a higher transcription level of argCJBDF in CO. The increased transcription level of argF might explain why replacement of the RBS and start codon could not lead to the desired l-ornithine production level. Therefore, we continued to insert a terminator (rrnB from plasmid pXMJ19) before argF to reduce the transcription level of argF, generating strain CO-6. Interestingly, after 48 h of shake flask fermentation, strain CO-6 produced 5.53 g/L of l-ornithine, which was 29.5% higher than the production level of the argF deletion strain CO-1 (4.27 g/L) . Compared with CO-1, the cell growth of CO-9 also improved from OD600 = 12.2 to OD600 = 13.5, after 48 h of incubation. The argF expression levels of the recombinant strains CO-8, CO-9, and CO-25, with terminator strengths of 239.91, 216.60, and 10.94, were 34, 35, and 65%, respectively, indicating that the expression level of argF decreased with improved terminator strength. In addition, insertion of a terminator in front of argF was an efficient strategy for improving l-ornithine production, by slightly relieving the growth disturbance, by blocking l-ornithine degradation. Moreover, these results also suggested that addition of a terminator could act as a reliable method for controlling gene expression and are a potential genetic engineering tool for C. glutamicum.In order to increase argF specifically here, was observed in the study on l-arginine auxotroph in engineered C. glutamicum S9114, which employed a common strategy used in the construction of engineered strains for l-ornithine production  enzyme activity, which reduced the yield of l-ornithine, consistent with the results observed in a previous study (Kim et al. C. glutamicum ATCC 21831; however, the cost of l-arginine addition and the genetic instability from using a plasmid hampered its industrial application in l-ornithine production. Thus, to address these problems, attenuation of the expression of argF, instead of direct gene deletion, was explored to balance l-ornithine production and cell growth. First, we chose an RBS optimization strategy, which is a promising metabolic engineering method that has been applied to construct a pathway for various compounds in previous reports (Sun et al. argF. However, replacement of the original RBS of argF with sequences with low translation initiation intensity could not achieve the desired l-ornithine production, though cell growth was unaffected.Based on our current knowledge, gene knockout is an effective strategy in genetic engineering for developing high target-compound producing strains. However, deletion of growth-coupled genes can induce some undesired results, such as a growth deficiency or extra nutritional requirements, which are undesirable for industrial fermentation. The same phenomenon of biomass deficiency caused by deletion, argCJBDFR operon in the l-arginine biosynthetic pathway (Chen et al. l-ornithine degradation. The transcription of argF plays a more important role in l-ornithine catabolism. In view of this, to reduce the transcription level of argF, transcription terminators, which are known to play critical roles in regulating natural genetic systems and implementing synthetic genetic logic, are employed (Cambray et al. argF deletion, l-ornithine production titer was improved by 29.5% after insertion of the rrnB terminator in front of argF. According to previous study, the terminators can not only stop the transcription process, but also function to prolong the mRNA half-life period, thus, stimulating the expression of upstream genes (Curran et al. argF causes high expression of the argCJBD gene cluster. Overexpression of the argCJBD operon is essential for l-ornithine biosynthesis (Hwang et al. C. glutamicum 1006ΔargR was reported to significantly improve l-ornithine fermentation (Hao et al. l-ornithine production was attributed to the activated expression of the upstream genes by the terminator. A similar conclusion had been drawn in a previous work, where integration of the transcription terminator downstream of the target genes caused a significant improvement of the expression level in a plasmid (Ito et al. We speculate that deletion of ArgR inactivated the feedback inhibition of the transcription of the argF, at both the translation and transcription levels, with the aim to increase the production of l-ornithine. Those strategies with several advantages over the previous argF deletion can be employed to the existing strains to further improvement of l-ornithine production and may be widely applicable to fine turning the expression of other growth coupled enzymes. To our knowledge, this is the first study in which a terminator-based strategy has been successfully developed to reduce the expression of argF and enhance l-ornithine production. The relative mRNA analysis provides valuable information for enhancing the expression of the upstream genes by insertion of terminator, which may find use in the construction of other middle metabolite producing strains. However, we failed to determine the optimal expression of argF for l-ornithine production without cell growth disturbance, and the l-ornithine production titer achieved in this study was relatively lower than that reported by other studies (Kim et al. l-ornithine production, which has potential application in engineering other high l-ornithine strains. The successful application of a terminator to regulate upstream and downstream gene expression also hinted at the possible applications of terminators in metabolic engineering to produce valuable products, which would enrich the metabolic engineering strategies for selective strain development. In our recent work, this strategy was employed for development of even higher l-ornithine producing strains by attenuation of proB and ncgl2228 Zhang et al. (C. glutamicum strains. We expect this technology to be extended to allow more laboratories to use it. In our next work, more experiments and examples based on terminators will be provided in the development of high l-ornithine producing strains.In conclusion, numerous efforts have been made to attenuate the expression of g et al. . AccordiAdditional file 1. Primers used in this study.


Background and objectives: The pathophysiology of tethered cord syndrome (TCS) in children is not well elucidated. An inelastic filum terminale (FT) is the main factor underlying the stretching of the spinal cord in TCS. Our study aimed to investigate the expression of glutathione-S-transferase (GST) in children and fetal FT samples in order to understand the relationship between this enzyme expression and the development of TCS. Materials and Methods: FT samples were obtained from ten children with TCS (Group 1) and histological and immunohistochemical examinations were performed. For comparison, FT samples from fifteen normal human fetuses (Group 2) were also analyzed using the same techniques. Statistical comparison was made using a Chi-square test. Results: Positive GST-sigma expression was detected in eight (80%) of 10 samples in Group 1. The positive GST-sigma expression was less frequent in nine (60%) of 15 samples from Group 2. No statistically significant difference was detected between the two groups (p = 0.197). Conclusions: Decreased FT elasticity in TCS may be associated with increased GST expression in FT. More prospective studies are needed to clarify the mechanism of the GST–TCS relationship in children. Tethered cord syndrome (TCS) is a functional disorder of the spinal cord which is secondary to stretching of the spinal cord. Impaired oxidative metabolism is the pathophysiological basis of TCS ,2,3,4,5.Fixation, stabilization, and buffering of the distal spinal cord from traction are the functions of the FT . This isGlutathione-S-transferase (GST) enzyme activity is present in the human body. GSTs are divided into different families: cytoplasmic, mitochondrial, or membrane-associated proteins involved in eicosanoid and glutathione metabolism ,18,19. ISigma-class GSTs belong to the cytosolic GST (cGST) family. These enzymes are widely distributed throughout nature . They noIn our study, we aimed to clarify the relationship between histologically abnormal FT and tethering of the spinal cord. Therefore, we investigated the expression of sigma-class GST in FT samples which were obtained from children who underwent surgery for TCS. In addition, we analyzed the expression of this enzyme in fetal FT samples and compared the results.Ethical approval for this study was obtained from the National Ethics Committee . Ten pediatric patients with a diagnosis of TCS underwent surgical treatment and cutting of the filum terminale and release of the spinal cord was performed in these patients (Group 1) .An FT sample was obtained from each patient for histological analysis A,B. We oIn terms of the gender of the fetuses, eleven were female and four were male. The mean gestation age was 25.4 weeks (range of between 14 and 35 weeks). There was no spinal malformation in the fetuses. No sign or findings of spina bifida were observed on the backs of the fetuses. A wide lumbosacral laminectomy was performed in the midline in order to reach the dural sac. Using microsurgical techniques, the conus medullaris and FT were seen after the dural opening. About 1 cm long segments of FT were determined and obtained for histological examination C. Generav/v) in methanol for 10 min at room temperature (RT) for immunohistochemical examination. Then, the sections were washed in distilled water for 5 min. In a domestic pressure cooker, antigen retrieval was performed for 3 min using 0.01 M citrate buffer (pH 6.0) . The sections were transferred into 0.05 M Tris-HCl (pH 7.6) containing 0.15 M sodium chloride (TBS) following washing in distilled water. These sections were incubated for 10 min at RT with super block (SHP125) . Then, they were covered with the primary antibody diluted 1:50 for GST Sigma (GSTS)  in TBS at 4 °C overnight. The sections were incubated at RT for biotinylated link antibody (SHP125)  following washing them in TBS for 15 min. Finally, treatment was followed with Streptavidin/HRP complex (SHP125) . In order to visualize peroxidase activity in the tissues, diaminobenzidine was utilized. Nuclei were lightly counterstained with haematoxyline. The sections were dehydrated and mounted after this procedure. Positive and negative controls were included in each run.FT samples were fixed in 10% buffered formalin. Then, they were embedded in paraffin blocks. Sections of 4 μm thickness were obtained. For the observation of general tissue morphology, one section was stained with H&E. Endogenous peroxidase activity was blocked by incubating the sections in 1% hydrogen peroxide , weak (+), moderate (++) or strong (+++). The results of sigma-class GST expression were compared with a Chi-square test for both groups. A Ten FT samples from children with TCS (Group 1) and 15 FT samples from normal human fetuses (Group 2) were evaluated with histopathological and immunochemical examinations. Fibrosis was observed in 8 samples in Group 2 , while ip = 0.197)  .Fibrosis and an inelastic structure of the FT were obvious in histological examination of ten children with TCS. In addition, sigma-class GST expression was frequent in FT samples from the children with TCS. Frequent expression of GST may contribute to the inelastic properties of FT in TCS. However, this difference was not statistically significant and this may be due to the low number of samples in each group.The filum terminale is an important fibrous anatomical structure in the pathophysiology and treatment of TCS. It is crucial to understand the morphometric parameters, histological structure, variants, and imaging of the FT for better diagnosis or treatment of TCS . The FT Glutathione S-transferases are multifunctional enzymes that can be found in prokaryotic and eukaryotic cells . Based oGSTs are also important in the cellular uptake and intracellular transport of bilirubin . Some isConnective tissues are mainly formed by fibroblasts which can be found throughout the body. These cells are the principal source of the extensive extracellular matrix (ECM). Fibrosis is the thickening of the ECM secondary to an inflammation or physical injury. It plays a significant role in organ failure . IdiopatThere are three limitations of this study. First of all, the number of samples in both groups is too low for more accurate conclusions to be drawn. Second is the lack of investigation of PGD2 to unveil the link between the development of fibrosis and the expression of GST. Third is the wide variation in the age of the patients with TCS; this variation may confound the results of GST expression, fibrosis, and structure of the FT. Studies with more FT samples and analysis of PGD2 may be helpful to better understand the correlation between fibrosis and TCS.Although statistically insignificant, expression of sigma-class GST is high in the filum terminale of children with TCS. This may be associated with decreased elasticity of FT. Further histological studies are needed to elucidate the pathophysiology of TCS.