Arabidopsis mutants of all these genes exhibit similar stress-sensitive phenotypes. RNA-seq analyses of these mutants detected significant intron retention in many stress-related genes under salt stress but not under normal conditions. Our study not only identified several novel regulators of pre-mRNA processing as important for plant stress response but also suggested that, in addition to RNAP II CTD that is a well-recognized platform for the recruitment of mRNA processing factors, FRY2/CPL1 may also recruit specific factors to regulate the co-transcriptional processing of certain transcripts to deal with environmental challenges.Eukaryotic genomes encode hundreds of RNA-binding proteins, yet the functions of most of these proteins are unknown. In a genetic study of stress signal transduction in Arabidopsis, we identified a K homology (KH)-domain RNA-binding protein, HOS5 (High Osmotic Stress Gene Expression 5), as required for stress gene regulation and stress tolerance. HOS5 was found to interact with FIERY2/RNA polymerase II (RNAP II) carboxyl terminal domain (CTD) phosphatase-like 1 (FRY2/CPL1) both in vitro and in vivo. This interaction is mediated by the first double-stranded RNA-binding domain of FRY2/CPL1 and the KH domains of HOS5. Interestingly, both HOS5 and FRY2/CPL1 also interact with two novel serine-arginine (SR)-rich splicing factors, RS40 and RS41, in nuclear speckles. Importantly, FRY2/CPL1 is required for the recruitment of HOS5. In  fry2 mutants, HOS5 was unable to be recruited to nuclear speckles but rather was mainly localized in the nucleoplasm. Mutants in these genes have similar stress-sensitive phenotypes. Transcriptome analyses identified significant intron retention in many stress-related genes in these mutants under salt stress conditions. Our study reveals that, in addition to RNAP II, the CTD phosphatase may also recruit specific splicing factors and RNA binding proteins to regulate the co-transcriptional processing of certain transcripts to deal with environmental stresses.Pre-mRNA processing, including 5′ capping, splicing, and 3′ polyadenylation, is critical for gene expression and is closely coupled with transcription. Phosphorylated carboxyl terminal domain (CTD) of RNA Polymerase II (RNAP II) serves as a platform for the recruitment of pre-mRNA processing factors, yet other components involved in the recruitment are less known. In a genetic study of stress signal transduction in Arabidopsis, we isolated a KH-domain RNA-binding protein HOS5 that plays important roles in stress gene regulation and stress tolerance. We found that HOS5 interacts with FIERY2/CTD phosphatase-like 1 (FRY2/CPL1) and they both also interact with two novel splicing factors, RS40 and RS41, in nuclear speckles. In  Pre-mRNA processing, including 5′ capping, splicing and 3′ end formation, is highly regulated and often coupled with transcription to increase its accuracy and efficiency. The carboxyl terminal domain (CTD) of Rpb1, the largest subunit of RNA polymerase II (RNAP II), serves as a platform for the recruitment and assembly of these processing factors Pre-mRNA splicing is an important step in mRNA maturation in eukaryotes. The accurate recognition and excision of introns are vital and require the coordinated function of a large number of proteins. Splicing takes place in the spliceosome, a large RNA-protein complex that includes 5 snRNAs and approximately 180 proteins There are several common types of AS including intron retention, exon skipping, and alternative 5′ splice site or 3′ splice site selection. Among these types, intron retention has been shown to be the most frequent type of AS in Arabidopsis and other plants SR30 has been shown to have intron retention in the atprmt5 mutant RS40 was detected in the upf1-5 and upf3-1 mutants or after CHX treatment SR45a has been observed under high light, salt, heat, and dehydration stress conditions One important group of splicing factors is the Serine/Arginine-rich protein group (SR proteins). These SR proteins are also involved in mRNA nuclear export, mRNA stability control, and genome maintenance RD29A::LUC (RD29A promoter driven the firefly LUCIFERASE gene) hos5-1 (high osmotic stress gene expression 5-1), that showed higher levels of LUC expression than the wild type under NaCl or the phytohormone abscisic acid (ABA) treatment, was isolated previously HOS5 encodes a KH-domain RNA-binding protein that was localized in the nuclear speckles. We also found that HOS5 interacts with two SR proteins and FIERY2/RNAP II CTD phosphatase-like 1 (FRY2/CPL1). Interestingly, the subnuclear localization of HOS5 depends on FRY2/CPL1. In fry2-1 mutants, HOS5 was no longer localized in nuclear speckles but rather mainly in the nucleoplasm. FRY2/CPL1 also interacts with the two SR splicing factors and all these mutants exhibited similar ABA- and salt-sensitive phenotypes as hos5. RNA-seq analyses revealed that many transcripts were misspliced with one or more introns retained in the mutants under salt stress conditions. The current study identified HOS5 as a novel factor involved in splicing and stress response. We proposed that FIERY2/CTD phosphatase-like 1 (FRY2/CPL1), like CTD itself, might help to recruit specific RNA processing factors for co-transcriptional processing of nascent transcripts to deal with environmental stresses.To understand gene regulation mechanisms under osmotic stress, we screened for Arabidopsis mutants with abnormal expression of the stress-inducible reporter gene hos5-1 mutant by screening for altered regulation of stress-responsive genes using the RD29A-LUC reporter RD29A-LUC line , hos5-1 mutant seedlings exhibited brighter luminescence after treatment with 100 µM ABA . Under NaCl treatments, the color of the leaves of hos5-1 was more yellowish than that of the wild-type seedlings, supporting the notion that the hos5-1 whole seedling was more sensitive to salt stress.We previously isolated the 0 µM ABA  or 300 m0 µM ABA . Quantit1 mutant . FurtherHOS5 gene, we generated a mapping population from a cross between hos5-1 and the Columbia-0 (Col-0) ecotype. Genetic mapping delimited the HOS5 locus to the BAC clone MNB8 on Chromosome 5. The genomic DNA in this region was amplified from the wild-type and hos5-1 mutant plants and sequenced. A single G to A mutation in the second exon of the At5g53060 gene was found. This gene encodes a KH-domain protein  plants . Compared with Col-0, the germination of hos5-2 mutant seeds   reporter gene were generated and GUS expression in independent lines was examined. GUS expression patterns (To determine the regulation of the R (qPCR) . HOS5 wapatterns  were simpatterns .To examine the subcellular localization of the HOS5 protein, stable transgenic Arabidopsis plants expressing HOS5 fused in frame with the green fluorescent protein (GFP) at the C-terminus were obtained. Several independent lines were examined for GFP expression. The GFP signals were detected in the nucleus of root cells by fluorescence microscopy . This nuThe splicing factor CypRS64 has been used as a marker for pre-mRNA splicing and it is localized in nuclear bodies and splicing speckles hos5-1hos5. To verify this interaction, we used FRY2 as bait to conduct another round of screens. We isolated six clones of HOS5, along with several other nuclear proteins (our unpublished data).In order to understand the molecular mechanisms of HOS5 in gene regulation, we investigated whether or not there are any proteins that interact with HOS5. We first conducted a yeast two-hybrid screen using HOS5 as bait. The screen identified two proteins that interact with HOS5. One turned out to be HOS5 itself, suggesting that HOS5 may form a homodimer (data not shown). The second HOS5-interacting protein was FIERY2/RNAP II CTD phosphatase-like 1 (FRY2/CPL1), which was identified in the same genetic screen as rd and 4th KH-domain could grow on -TLHA medium, whereas those lacking these two domains or only having parts of them could not grow, indicating that the region harboring the 3rd and 4th KH-domain of HOS5 is responsible for interacting with FRY2. Interestingly, the hos5-1 mutation did not affect the HOS5-FRY2 interaction (hos5-1 mutation did not affect HOS5 dimerization either (To map the interacting domain(s) of FRY2 and HOS5, we constructed full-length HOS5 in the pBD vector and full-length and various truncated FRY2 into the pAD vector. As shown in eraction . Furthern either .2 beads using standard protocols. The protein complexes bound with the beads were eluted with 3× FLAG peptide. The eluted proteins were then subjected to trypsin digestion and mass spectrometry analysis. We identified four peptide fragments that matched to an uncharacterized SR protein RS41  assays. Protein extracts from seedlings of the Col-0 (control) and the HOS5-FLAG-tagged lines were subjected to Co-IP with anti-FLAG Mein RS41 . This prein RS41 . In Arabein RS41 . It is tN. benthamiana) leaves by Agrobacterium-mediated leaf disc transformation. Co-IP was carried out by using anti-FLAG antibody for the pull down and normal IgG as a control. Samples were subjected to SDS-PAGE and western blot analysis. We found that both RS40 and RS41 could be pulled down by anti-FLAG antibody  assays in Arabidopsis protoplasts.  control . As expe control  but does control . Meanwhifry2 mutants are similarly sensitive to stress like hos5 , we hypothesized that the null mutants of RS40 and RS41 may also have similar phenotypes as the hos5 mutants. We obtained two T-DNA insertion lines, WiscDsLox382G12 (named as rs40-1) and SAIL-64-C03 (named as rs41-1)  of rs40-1 and rs41-1 seeds was more strongly inhibited by ABA, particularly in the early stages. After germination, all cotyledons of Col-0 seedlings became green, whereas all cotyledons of hos5-2 were yellow. On the other hand, approximately 50% of the cotyledons of rs40-1 and rs41-1 were yellow  . The exp rs41-1) . These me yellow . We alsot stress . Furtherhos5, fry2-1 and the two rs mutants. Leaf samples from seedlings without or with NaCl treatment were fixed by formaldehyde and hybridized with an oligo-dT probe labeled with Alexa Fluor 488. As shown in hos5-1 under normal conditions. When treated with NaCl or ABA, both the wild type and the mutants had an increased accumulation of polyA RNA in the nuclei. Nonetheless, hos5-1 and to a lesser extent, fry2-1, accumulated more polyA mRNA in the nuclei than did the wild type, especially under NaCl treatment  between the wild type and hos5-1 mutant. We generated 359 million single-end/paired-end reads (101 bp in length), respectively. On average, about 90% of these reads could be unambiguously aligned to the TAIR10 reference genome sequence (fragments per kilobase of exons per million mapped reads - FPKM>0.85). Comparison of the mapped reads against the gene model (version TAIR10) revealed that ∼95% of the reads mapped to the exonic regions, whereas only ∼3% mapped to intergenic regions. Plotting the coverage of reads along each transcribed unit exhibited a uniform distribution, with no obvious 3′/5′ bias, which reflects a high quality of the cDNA library (data not shown). Furthermore, assessing the sequencing saturation showed that as more reads were obtained, the number of newly discovered genes plateaued  using the Illumina Hi-Seq platform to examine the global pre-mRNA splicing in the wild type and mutants under salt stress. These mutants included the respective single mutants as well as lateaued , suggesthos5-1 mutants, 57 intron retention events  were represented. In fry2-1, hos5-1 fry2-1 double mutants and rs40-1 rs41-1 double mutants, the intron retention events were 247, 586, and 47, respectively. Interestingly, Gene Ontology (GO) analysis of the genes undergoing aberrant splicing in the above mutants revealed a striking enrichment in the response-to-abiotic-stress categories (hos5-1 and fry2-1 (2+/H+ exchanger CAX1 (AT2G38170), which is activated by the SOS2 kinase and links Ca2+ with salt tolerance RCI2A, a cold-inducible gene that also enhances salt tolerance hos5-1, fry2-1, and hos5-1 fry2-1 double mutants. For instance, half of alternative splicing events in hos5-1 were also found in the fry2-1 mutant, which is statistically very significant compared with those from random sampling of annotated introns   .hos5-1 and fry2-1, respectively. hos5-1, fry2-1, and hos5-1 fry2-1 double mutants. Validation for intron retention in rs40-1 rs41-1 double mutants was presented in We selected six genes with intron retention events and validated them by RT-PCR using intron-flanking primers. fry2-1 or hos5-1 mutant seedlings. Whereas the localization of the FRY2 protein in the hos5-1 mutant was not altered (data not shown), we found that most of the HOS5 protein was mislocalized in the fry2-1 mutant. As shown in fry2-1 mutant. These included the nucleoplasm localization (but absent from the nucleolus) in 28% of the protoplast cells and mostly diffused localization in the nucleus with accumulation in a few nuclear speckles in 51% of the protoplast cells of the fry2-1 mutant. Only in 21% of the protoplast cells of the fry2-1 mutant was the HOS5 protein correctly localized in nuclear speckles. Overall, these data reveal that FRY2 is essential for the recruitment of HOS5 to nuclear speckles and presumably this will also affect HOS5's role in pre-mRNA processing.The specific localization of HOS5 and FRY2 proteins in the nucleus prompted us to ask how these proteins are recruited to their particular subnuclear location, namely, nuclear speckles. We transiently expressed the respective GFP-tagged proteins in leaf protoplasts prepared from the wild type, hos5 mutants are more sensitive to osmotic stress . In this study, we identified HOS5 as a KH-domain protein. The K homology (KH) domain was originally identified in the heterogeneous nuclear ribonucleoprotein K (hnRNP K) of humans but it is also found in various proteins in archaea, bacteria and other eukaryotes atprmt5 mutant HOS5 is a negative regulator of osmotic stress-responsive gene expression hos5-1 mutant has a mutation in the conserved glycine residue in the second KH domain, which likely affects the ability of HOS5 to bind its RNA targets. We attempted to isolate the RNA targets, but perhaps due to their low abundance, we could not identify with confidence the RNA species that are specifically bound by HOS5. However, through the identification of protein partners, we were able to uncover the molecular functions and modes of action of HOS5. In yeast two-hybrid screens, we identified the Pol II CTD phosphatase-like FRY2/CPL1 as an interacting partner. It has been demonstrated that FRY2/CPL1 can dephosphorylate Ser-5 but not Ser-2 of phosphorylated CTD hos5-1RD29A::LUC in fry2-1 is slightly higher than that in hos5-1, we indeed detected clear impairments in pre-mRNA splicing in these mutants under salt stress conditions. We found that intron retention is common among the mutants, with the retention in hos5 and fry2 mutants have defects in pre-mRNA splicing and that HOS5 and FRY2/CPL1 both interact with the splicing factors RS40 and RS41 in nuclear speckles is consistent with the idea that these proteins are involved in splicing of pre-mRNA. Nuclear speckles are interchromatin domains where many splicing factors, including snRNP and SR proteins, are stored and modified to be supplied to active transcription sites and thus are closely associated with gene transcription The fact that both fry2-1 mutant, HOS5 protein could not accurately be recruited to nuclear speckles as in the wild type plants . cDNAs were synthesized from total RNA by using Superscript III reverse transcriptase (Invitrogen). Real-time quantitative PCR was performed on the ABI 7900HT Fast Real-Time PCR System using HOS5 promoter (1.8 Kb) was amplified from genomic DNA and ligated to pENTR-D-TOPO. The promoter was cloned into the destination vector pMDC162 Agrobacterium-mediated flower dipping. For the β-glucuronidase (GUS) analysis, plant samples were stained in GUS staining buffer at 37°C overnight followed by destaining with 75% ethanol.For co-localization assays, HOS5, CypRS64, FRY2, RS40 and RS41 were cloned into pENTR-D-TOPO and transferred to destination vectors containing EGFP or mRFP respectively by LR reaction. Plant seedlings and protoplasts expressing these fused proteins were imaged with an LSM 710 inverted confocal microscope .Standard Co-IP protocol was performed. Briefly, 2 g of leaves of HOS5-FLAG plants were grounded and lysis buffer  was added to each sample. After centrifugation, the supernatant was transferred to a new tube. The samples were pre-cleaned by adding normal IgG-conjugated beads. The M2 beads were then added into the samples and incubated for 3 hr. Afterwards, the beads were washed 5 times with the lysis buffer and twice with TBS buffer. Proteins were eluted by adding 3×FLAG solution and were then subjected to SDS-PAGE and in-gel digested for mass spectrometry analysis.Yeast transformation was performed according to the manufacturer's instructions . To map the interacting domain, FRY2 and HOS5 deletion constructs were made from both the N- and C-termini. These deletion mutants were then tested for interaction by co-transforming them into yeast strain AH109 with the corresponding full-length partner (FRY2 in pGAD or HOS5 in pGBK) and plating on -tryptophan/-leucine  and -tryptophan/-leucine/-histidine/-adenine  media supplemented with X-α-Gal. For FRY2, seven deletion constructs were tested. For the HOS5, eight deletion constructs and the hos5-1 mutant were tested.Poly-A RNA in situ hybridization was performed as described http://www.arabidopsis.org/). The raw reads were aligned to genome sequences using TopHat (2.0.7) http://www.r-project.org/) was performed to identify differential representation of introns in wild type and mutants using read coverage from an intron and the corresponding exons. Introns with more than 10 times coverage and two-sided P values less than 0.01 were regarded as the intron retention events. Primers were designed according to RNA-seq results with one primer in a primer pair covering the exon-exon junction Using the TRIzol Reagent , total RNA was extracted from 12-day-old seedlings treated with 300 mM NaCl for 3 hr. Polyadenylated RNA was isolated using the Oligotex mRNA Midi Kit . The RNA-seq libraries were constructed using an Illumina Whole Transcriptome Analysis Kit following the standard protocol  and sequenced on the HiSeq platform to generate high-quality paired-end reads. For RNA-seq data analysis, Arabidopsis genome sequences and annotated gene models were downloaded from TAIR10  Luminescence of the wild-type (left) and hos5-1 (right) seedlings without treatment. (B) Luminescence after 100 µM ABA treatment for 3 hr. (C) Luminescence after 300 mM NaCl treatment for 3 hr. (D) LUC expression levels under different conditions measured by Real-Time Quantitative RT-PCR. ACTIN2 was used as an internal control. Error bars represent the standard deviations (n = 3). (E) Seed germination under different concentrations of ABA. Seeds were surface-sterilized and planted on ½ MS agar media supplemented with the indicated concentrations of ABA and incubated at 4°C for 3 days before being placed at 22°C for germination. Germination (radicle emergence) was scored 3 days later. Results are means and standard errors from three replicates with 100 seeds for each treatment. (F) ABA sensitivity of hos5-1 mutant seedlings. Seven-day-old hos5-1 and C24 seedlings were transferred from ½ MS-agar media to media containing the indicated concentrations of ABA. The photos were taken 2 weeks after the transfer. (G) Root growth of hos5-1 under salt stress. Four-day-old seedlings of C24 and hos5-1 were transferred from ½ MS agar plates to plates supplemented with 120 mM NaCl. Root elongation was measured 7 days after the transfer. Results are means and standard errors (n = 12). (H) Seed germination sensitivity to salt stress. Seeds were planted on ½ MS medium plates with 100 mM NaCl and vernalized at 4°C for 3 days before being placed at 22°C for germination. Germination was then scored daily for 6 consecutive days. (I) hos5-1 seedlings were slightly more sensitive to salt stress than C24 seedlings. Seven-day-old C24 and hos5-1 seedlings were transferred from ½ MS-agar medium plates to ½ MS-agar plates supplemented with 0, 60, or 120 mM NaCl. The photos were taken 3 weeks after the transfer.The luminescence and stress phenotypes of the (TIF)Click here for additional data file.Figure S2nd KH domain in KH domain-containing proteins of Arabidopsis. The red star indicates the most conserved glycine residue in the 2nd KH domain, which was mutated in hos5-1.HOS5 structure and sequence comparison with other homologous proteins. (A) 3D-structure of HOS5 predicted by Phyre2. KH domains are alpha helixes and KH1 through KH5 are in blue, green, yellow, brown and red, respectively. (B) The cladogram analysis of all multi-KH domain-containing proteins in Arabidopsis. (C) Alignment of the 2(TIF)Click here for additional data file.Figure S3hos5-2 has similar phenotypes as hos5-1. (A) Structure of the HOS5 gene and the locations of hos5-2 and hos5-3 mutations. Black boxes, exons; white boxes, untranslated regions (UTRs); lines, introns; star, hos5-1 mutation site; black triangles, T-DNA insertion sites. (B) RT-PCR analysis was performed to check the expression level of HOS5 in Col-0, hos5-2 and hos5-3 mutants. ACTIN 2 was used as control. This experiment was repeated 3 times and the same result was obtained. (C) The phenotype of hos5-2 under salt stress. Four-day-old seedlings of Col-0 and hos5-2 were transferred on to vertical ½ MS agar plates without (left panel) or with (right panel) 120 mM NaCl. The pictures were taken 7 days after the transfer. (D) Root elongation of Col-0 and hos5-2 under salt stress as shown in (C). Results are means and standard errors (n = 12). (E) Seed germination and early seedling development under ABA treatment. Seeds of Col-0 and hos5-2 were surfaced sterilized and planted on ½ MS agar plates with 2.0 µM ABA. After 3-day vernalization, the plates were placed at 22°C for germination and growth. The picture was taken 7 days after the plate being incubated at the room temperature. (F) Relative root elongation of hos5-2. Four-day-old seedlings of Col-0 and hos5-2 were transferred to ½ MS agar plates with 15 µM ABA. The root elongation was measured 7 days after the transfer. Results are means and standard errors (n = 12). Blue, Col-0; green, hos5-2.(TIF)Click here for additional data file.Figure S4Negative controls of the BiFC assays. HOS5, FRY2, RS40 and RS41 were respectively fused to EYFP-N. HAP5C, a transcription factor used as negative control, was fused to EYFP-C. E-YN and E-YC are empty vectors. The combinations of plasmids were transformed into Arabidopsis protoplasts as indicated. Shown are merged bright field and fluorescence images .(TIF)Click here for additional data file.Figure S5RD29A-LUC expression in hos5-1 fry2-1 double mutants. (A) Luminescence images of C24, hos5-1, fry2-1 and hos5-1 fry2-1 double mutants without (CK) or with 300 mM NaCl treatment for 3 hr. T2–3, T2–6 and T2–22 are 3 lines of the double mutant. (B) Quantification of luminescence intensity in (A). Error bars represent standard deviation (n = 15). Blue, control treatment (CK); orange, NaCl treatment.(TIF)Click here for additional data file.Figure S6RS40 and RS41 genes. Black boxes, exons; white boxes, UTRs; lines, introns; black triangles, T-DNA insertion sites. (D) Real-time qPCR analysis of the expression levels of RS40 and RS41 in Col-0, rs40-1 and rs41-1 mutants, respectively. ACTIN 2 was used as a control. The experiment was repeated 3 times and similar results were obtained each time. (E) Seeds germination on ½ MS agar plates containing 2.0 µM ABA. Seeds were surface sterilized and incubated at 4°C for 3 days before being placed at 22°C for germination. The germination rates were scored daily for 7 consecutive days. Results are means and standard errors (n = 3). (F) Sensitivity of RS40 and RS41 seed germination to ABA treatment. The wild-type and mutant seeds were allowed to germinate and grow on 2.0 µM ABA plates. The picture was taken 7 days after germination. (G) Four-day-old seedlings of Col-0, hos5-2, rs40-1 and rs41-1 were transferred from ½ MS agar plates to ½ MS agar plates supplemented with 120 mM NaCl. Root elongation was measured. Results are means and standard errors (n = 12). Blue, Col-0; orange, hos5-2; green, rs40-1; purple, rs41-1. (H) and (I), Expression of the RS40 and RS41 genes under stress condition. ACT2 was used as an internal control in qPCR assays. Error bars represent the standard deviations (n = 3).RS40 and RS41 sequence characters and the stress phenotypes of their mutants. (A) The cladogram analysis of all SR splicing factor proteins in Arabidopsis. RS40 and RS41 are clustered together as indicated in a red rectangle. (B) Sequence alignment of RS40 and RS41. The red lines indicate the peptide fragments identified by mass spectrometry. All 4 fragments are in the RRM motif. (C) Schematic structure of the (TIF)Click here for additional data file.Figure S7hos5-1 RNA-seq data and Gene Ontology of splicing-defective genes. (A) Randomly sampled reads were plotted against the mapped genes for the wild type and mutants. x-Axis shows the number of the mapped reads and y-axis displays the number of the expressed genes. (B) Gene Ontology of the genes with splicing defects in mutants.The properties of (TIF)Click here for additional data file.Figure S8hos5, the hos5-1 mutant; fry2, the fry2-1 mutant; hos5/fry2, the hos5-1 fry2-1 double mutant. The list of genes with splicing defects in these mutants can be found in A Venn diagram showing the number of genes with intron retention common among different mutants. (TIF)Click here for additional data file.Table S1List of primers used in this study.(XLSX)Click here for additional data file.Table S2hos5-1 mutant.List of genes with intron retention in the (XLSX)Click here for additional data file.Table S3fry2-1 mutant.List of genes with intron retention in the (XLSX)Click here for additional data file.Table S4hos5-1 fry2-1 double mutant.List of genes with intron retention in the (XLSX)Click here for additional data file.


Acoustic trauma is an injury to the hearing mechanisms in the inner ear due to excessive noise. This injury is the most prevalent cause of sensorineural hearing loss in humans, especially from occupational exposure. Previous studies have shown the essential role of free radical formation in the inner ear hearing loss caused by acoustic trauma.This study was performed to determine the effect of N-acetylcysteine (NAC) administration for reducing acute acoustic trauma in rabbits.Twenty four rabbits were assigned to four groups including: control, noise plus saline, noise plus NAC administration , three days before exposure to noise and three days after noise exposure), and NAC alone. Auditory brain stem response (ABR) threshold was measured before exposure and one hour and 14 days after exposure.The saline plus noise group had on average a 49 decibel (dB) temporary threshold shift (TTS) and 23.9 dB permanent threshold shift (PTS) at the studied frequencies, while rabbits in the NAC administration plus noise group had a 31.5 dB TTS and 10.7 dB PTS averaged across the frequencies.Administration of NAC can provide appropriate protection against acoustic trauma-induced hearing loss in rabbits at all studied frequencies. Acoustic trauma is injury to the hearing mechanisms of the inner ear due to excessive noise > 85 dB). Acoustic trauma is a prevalent cause of sensorinural hearing loss and it may be caused by an explosion close to the ear, gun shots, long–term exposure to loud noise (such as machinery noise) -3. In ad dB. AcouThis study was conducted to determine the effect of N-acetylcysteine (NAC) administration on reducing temporary and permanent injuries resulting from acute acoustic trauma (excessive noise for short time exposure) in rabbits.Twenty four healthy, male, adult white New Zealand rabbits (2200 – 2500 g body weight) were obtained from the Pasture Institute of Iran. The rabbits were accommodated in separate cages and maintained on a light–dark cycle (12 – 12 h) at 23-27°C, with free access to food. All procedures relating to the care, use and handling of the rabbits were conducted in accordance with the principles of the Helsinki Protocol. The rabbits were divided randomly into four groups  according to the type of experimental condition as follows: Group 1, control (no exposure to noise and no injection); Group 2, exposed to noise (120 dB octave band noise centered at 4 kHz for four hours) and received saline intraperitoneal injection (IP); Group 3, exposed to noise (as in Group 2) and received NAC 325 mg/kg body weight IP injection; and Group 4, not exposed to noise and received NAC (325 mg/kg body weight IP injection). NAC or saline was injected IP at a single dose (325 mg/kg body weight) once a day from three days before noise exposure to three days after noise exposure . NAC was administrated 325 mg/kg body weight because previous studies had shown that this dose was the most effective for the reduction of continuous noise induced hearing loss . Groups Auditory brain stem response (ABR) was used to evaluate the rabbits’ auditory function. ABR is based on the recording of auditory evoked potentials and they are widely used to evaluate levels of acoustic trauma-induced hearing loss in animal models , 13, 17.The Kolmogorov-Smirnov test (K-S test) was used to evaluate the normality of data in all groups. One-way analysis of variance (ANOVA) was used to compare ABR threshold shifts among groups at the study frequencies. Tukey’s test was applied as a post-hoc test for multiple comparisons among the groups. Latency time of wave V before and after noise exposure was compared using a paired t-test at each frequency in all groups. A P value smaller than 0.05 was considered significant.Both ABR threshold and latency time of wave V before exposure (baseline) at each frequency were equal in all the experimental groups. The latency time wave V at 8 and 4 kHZ were significantly lower than 2 and 1 kHZ in all groups. The latency time wave V before exposure and one hour after exposure at each frequency in the experimental groups are represented in The purpose of the study was to investigate the preventive effects of NAC administration on acoustic trauma induced hearing loss in rabbits. Our results showed that when NAC was administered three days before acoustic trauma and three days after exposure to noise , an appropriate prevention was provided against acute acoustic trauma induced temporary and permanent hearing loss in rabbits. Acoustic trauma can cause reactive oxygen species (ROS) formation in the cochlea as a normal cellular byproduct. In a normal status, various natural antioxidant defense mechanisms may prevent these damaging free radicals from causing any permanent injury, but when acute acoustic trauma occurs, normal antioxidant defense mechanisms are no longer maintained and hair cells in the cochlea may undergo a non-recoverable injury leading to permanent hearing loss. In this situation, our results suggest that temporary and permanent hearing loss may be prevented or reduced by the administration of an antioxidant agent (NAC). This improvement may be due to glutathione (GSH) synthesis by NAC. GSH may also be able to recover the effects of glutamate exciototoxicity as well as protect cochlear mitochondria from free radical formation , 15. NAC


Sudden sensorineural hearing loss (SSNHL) is a debilitating condition with an incidence of nearly 20 per 100,000 in populations. Metronidazole-induced ototoxicity is an extremely rare etiology of SSNHL. In this report, we describe a young female with bilateral SSNHL due to oral use of metronidazole. A 23 years old female presented to the emergency department with acute bilateral hearing loss. We found out that her hearing loss had started 4 days after initiation of metronidazole which was administered for treatment of diarrhea. This case report shows that physicians should be aware of the uncommon side effects while prescribing metronidazole to patients in order to manage the possible adverse events on time. Sudden sensorineural hearing loss (SSNHL) is a subset of sudden hearing loss (SHL) which affects nearly to 20 per 100,000 in populations . Thus, dA 23 years old female presented to the emergency department with acute bilateral hearing loss. Her problem had first started 3 days ago by hearing a non-pulsatile sound and tinnitus in both ears. Her hearing had been progressively decreasing and worsened markedly within the next 3 days. She denied any ear pain and drainage, headache, vertigo or oral lesion and did not have fever and chills. There was no history of antecedent fluctuating hearing loss, head trauma, acoustic trauma, allergic rhinitis, sinusitis or recurrent urinary tract infection. She reported acute watery diarrhea in the past week. She had history of a rhinoplasty surgery 9 months previously. Initial history revealed no regular medication use. The patient did not smoke and did not use alcohol or illicit substances.The patient was alert and nervous. She could not hear verbal communication clearly, thus the physician used handwritten notes to communicate with the patient and vice versa. Her vitals were as follows: body temperature was 37.4°C, blood pressure was 110/65 mm Hg, heart rate was 100 beats per minute, and her respiratory rate was 18 breaths per minute. General physical examination was normal except for mild tenderness of both feet to light touch. The otoscopic examination of both ear canals and tympanic membranes were normal. There was no tragus tenderness. She had negative whisper sign on both sides. A complete neurologic examination was done and was normal.The patient was admitted to the ear, nose and throat ward with impression of sudden bilateral sensorineural hearing loss ematologic lab tests and biochemistry were done and all parameters were in normal range. She was negative for rheumatoid factor and had normal erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) levels. Audiometry of both ears confirmed SNHL (30-dB hearing loss at 3 consecutive frequencies).The initial empiric treatment was started immediately and included: bilateral intratympanic dexamethasone, oral prednisolone and oral acyclovir. There was no significant improvement by day 4 of admission. Due to increased foot pain and tenderness, the patient was admitted to the rheumatology ward for further investigation. Serologic studies for known autoimmune diseases were normal and viral markers were negative. Methotrexate, pentoxifylline and folic acid were added to the aforementioned treatment regimen. Ten days after treatment initiation, gradual improvement of hearing was reported by the patient. Also, serial audiogram at 10th day showed significant improvement especially in the right ear at low frequencies.We continued to search for the etiology of SSNHL in the patient by interviewing her and reviewing the history several times on different occasions. Serendipitously, we found out that her hearing loss had started 4 days after initiation of metronidazole which was administered for treatment of diarrhea.In this report we present an extremely rare etiology of bilateral SSNHL. There are only three metronidazole-associated SSNHL reports in the literature . MetroniThe patient described in this report presented to the Emergency Department with bilateral SHL. Audiogram confirmed the diagnosis of SSNHL. Despite complete physical examination and laboratory studies, the underlying cause remained unknown until we interviewed the patient several times and discovered metronidazole use. Early treatment and supportive care eventually reversed the SNHL although not completely. After 2 weeks of hospitalization, she was eventually discharged with oral folic acid, pentoxifyllin and calcium supplements.Recently a systematic review found no association between dose or duration of metronidazole use and rare side effects . In our Taken all together, this case suggests that physicians should be aware of the uncommon side effects while prescribing metronidazole to patients in order to manage the possible adverse events on time.Written informed consent was obtained from the patient.CRP: C-reactive protein; ESR: Erythrocyte sedimentation rate; SSNHL: Sudden sensorineural hearing loss.No competing interest exists for any of the authors associated with the manuscript.SMRH and SA drafted the manuscript. AJ and SMRH: contributed to acquisition of data and, interpreted the data. All authors read and approved the final manuscript.


Hearing thresholds of fishes are typically acquired under laboratory conditions. This does not reflect the situation in natural habitats, where ambient noise may mask their hearing sensitivities. In the current study we investigate hearing in terms of sound pressure (SPL) and particle acceleration levels (PAL) of two cichlid species within the naturally occurring range of noise levels. This enabled us to determine whether species with and without hearing specializations are differently affected by noise.Etroplus maculatus, which possesses anterior swim bladder extensions, and the slender lionhead cichlid Steatocranus tinanti, in which the swim bladder is much smaller and lacks extensions. E. maculatus was tested between 0.2 and 3kHz and S. tinanti between 0.1 and 0.5 kHz using the auditory evoked potential (AEP) recording technique. In both species, SPL and PAL audiograms were determined in the presence of quiet laboratory conditions (baseline) and continuous white noise of 110 and 130 dB RMS. Baseline thresholds showed greatest hearing sensitivity around 0.5 kHz (SPL) and 0.2 kHz (PAL) in E. maculatus and 0.2 kHz in S. tinanti. White noise of 110 dB elevated the thresholds by 0–11 dB (SPL) and 7–11 dB (PAL) in E. maculatus and by 1–2 dB (SPL) and by 1–4 dB (PAL) in S. tinanti. White noise of 130 dB elevated hearing thresholds by 13–29 dB (SPL) and 26–32 dB (PAL) in E. maculatus and 6–16 dB (SPL) and 6–19 dB (PAL) in S. tinanti.We investigated auditory sensitivities in the orange chromide Our data showed for the first time for SPL and PAL thresholds that the specialized species was masked by different noise regimes at almost all frequencies, whereas the non-specialized species was much less affected. This indicates that noise can limit sound detection and acoustic orientation differently within a single fish family. The auditory sensitivity of fishes has been measured in more than 150 species covering a large number of families, hearing sensitivities and habitats. Fay (1988) The detection of signals is impaired in the presence of other signals such as noise of a certain level – a phenomenon termed masking. Elevated auditory thresholds due to masking have been demonstrated in several fish species and in the presence of various noise types using different techniques . This has been shown using white noise as a masker in otophysines, gadids, batrachoidids, holocentrids, haemulids and centrarchids Wysocki and Ladich (2005a) Steatocranus tinanti (subfamily Pseudocrenilabrinae) has a reduced swim bladder without any connection to the inner ear. In contrast, the orange chromide Etroplus maculatus (subfamily Etroplinae) possesses anterior swim bladder extensions touching the cranium at the inner ears Previous studies chose hearing specialized and non-specialized species from non-related taxa such as otophysines and perciforms for comparative purposes to study masking. Investigating the effect of different maskers on different hearing sensitivities calls for choosing closely related species, ideally belonging to the same family, that differ considerably in their auditory sensitivities. An intra-familial masking study was carried out only within sciaenids (drums and croakers); it compared two species which, however, did not differ in absolute hearing sensitivities except in the maximum frequency detectable S. tinanti and E. maculatus, with special focus on characterizing the hearing thresholds in terms of sound pressure (SPL) as well as particle acceleration levels (PAL). We chose white noise at two different levels within the naturally occurring range as maskers  and in four specimens of S. tinanti . Fishes originated from local fish suppliers and were transferred to the University of Vienna in August/September 2011 and January 2012. Animals were kept in 98- and 245-l aquaria, which were equipped with a sand bottom, halved flower pots and  plants as hiding places. Water was maintained using external filters. Fishes were kept under a 12∶12 h L:D cycle at 25±1°C and were fed once daily with commercial flake food and red blood worms.We measured auditory sensitivities in six specimens of All hearing experiments were performed with the permission of the Austrian Federal Ministry of Science and Research (permit number GZ 66.006/0023-II/10b/2008).Auditory thresholds were determined by applying the AEP recording technique –1 body weight for S. tinanti and E. maculatus, respectively. All auditory measurements were carried out in an bowl-shaped plastic tub , which was lined inside with acoustically absorbent material (air-filled packing wrap) to minimize distortions of stimuli  at mean concentrations of 4.0 or 14.9 µg*gS. tinanti lacks hearing specializations (in contrast to E. maculatus) and we therefore assumed that it is mainly sensitive to particle acceleration In addition to SPL, we determined PAL at thresholds because 2 and −173.7 dB re 1 V/µPa) was placed instead of the fish at the position of the test subject in the tub. PALs at all stimulus frequencies and all noise conditions at hearing threshold levels of the fish were determined with the acceleration sensor oriented in vertical direction . Control measurements in all three directions  revealed that the contribution of other directions to the combined PAL was <1 dB  sensor  to obtain a flat noise spectrum underwater and fed to the second channel of a signal mixer (SM5 of TDT System 3)  averaged over 1 min measuring time. The Leq is a measure of the averaged energy in a varying sound level and commonly used to assess environmental noise. The system was calibrated using a Brüel & Kjaer 4229 calibrator. The LLeqs of the noise masker were 110 and 130 dB re 1 µPa. In addition, background noise levels in the experimental test tank  were measured. After SPL measurements, the background noise and the white masking noise were recorded via an external sound card  on a PC. Recording and analyzing were done using S_Tools-STX 3.7.8, an acoustics, speech, and signal processing application developed by the Acoustics Research Institute at the Austrian Academy of Sciences. Sound spectra of 1 min recordings were calculated by an FFT analysis using a filter bandwidth of 1 Hz. Absolute spectral values were calculated from the relative spectral values after calibrating the sound recording system  using the calibrator (Brüel & Kjaer 4229).The SPLs of the masking noise were measured at the position of the fish using a sound level meter (Brüel & Kjaer 2238 Mediator) connected via a hydrophone power supply (Brüel & Kjaer 2804) to the hydrophone (Brüel & Kjaer 8101). We determined L-weighted (5 Hz to 20 kHz) equivalent continuous SPL . Absolute spectral PALs were calculated from the relative values using the sensor sensitivity and the calibration factor of the sound recording system.As the assumption of normal distribution was met, parametric tests were applied. To determine if the presentation of masking noise resulted in significant threshold shifts between different noise conditions  repeated measures ANOVA was calculated at each frequency followed by LSD post hoc tests.E. maculatus revealed greatest hearing sensitivity between 0.2 and 1 kHz, with a steep decrease at higher frequencies (threshold at 500 Hz: 71.3 dB re 1 µPa)  . At a maquencies . At a noquencies , 3A (Tabquencies .E. maculatus showed highest sensitivity at 200 Hz (33. 6 dB re 1 µm/s2) and a significant decrease in hearing sensitivity at both masking noise levels at all frequencies measured   . In contly 16 dB , 5A (Tably 16 dB .2) (The baseline PAL-curve showed highest auditory sensitivity at 0.2 kHz (PAL: 48.4 dB re 1 µm/s2) . Again, 2) . Exposur2)  Table 12 (Table The large diversity in hearing sensitivities even among closely related fish species (holocentrids and cichlids) raises the question of the functional significance of this diversity. Masking studies such as the present one help to elucidate this diversity, which was first recognized in the 1930s As discussed by Ladich It is likely that eco-acoustical constraints, namely low background noise levels, resulted in the evolution of hearing enhancements, enabling fish species with such enhancements to detect low level sounds S. tinanti live in waters that are noisier than stagnant ones E. maculatus inhabits more stagnant and thus more quiet waters and will be able to use its improved hearing abilities.Rheophilic species such as the cichlid E. maculatus, hearing thresholds were significantly masked at their most sensitive frequencies (up to 1 kHz) at the lower masker level (SPL and PAL). Masking noise of 110 dB did not result in masking effects at 2 and 3 kHz because this species is approximately 30 dB less sensitive at these frequencies. A 20 dB increase in white noise (110 vs. 130 dB) increased the masked hearing thresholds linearly at the most sensitive frequencies. In contrast, S. tinanti was not affected by low levels of white noise at any frequency. At the higher noise level, hearing in S. tinanti was masked at the most sensitive frequencies . The loss in auditory sensitivity at 130 dB as compared to baseline levels is much smaller in S. tinanti than in E. maculatus.The current study reveals that white noise presented at levels typically encountered in natural habitats affects hearing in different representatives of the cichlid family differently, and that this effect depends on absolute hearing sensitivities. In A comparison of SPL and PAL threshold shifts reveals similar trends for both acoustic variables. This indicates that masking data based solely on SPL thresholds in prior studies on non-specialized species have a certain validity.Pogonias chromis and the Atlantic croaker Micropogonias undulatus – to two different levels of white noise (124 and 136 dB). The swim bladder of the black drum has no anterior projections, while that of the Atlantic croaker has extensions that approach the inner ears. The two species did not differ in absolute hearing sensitivities, indicating that the Atlantic croaker represents an intermediate form between fish with non-specialized and those with specialized swim bladders In our intra-familial masking study, specialized and non-specialized species distinctly differed in the masking effect. Ramcharitar and Popper (2004) Carassius auratus and the striped Raphael catfish Platydoras armatulus, which have similar baseline sensitivities (approximately 70 dB at 500 Hz) but belong to different orders within otophysines, a 20 dB increase in white noise level (110 dB vs. 130 dB) resulted in a threshold shift of approximately 20 dB at the most sensitive hearing range between 0.2 and 2 kHz E. maculatus, which is specialized for hearing similar to otophysines due to anterior swim bladder extensions, a similar shift in thresholds of approximately 20 dB was observed between 0.2 and 1 kHz. The Atlantic croaker possesses anterior swim bladder extensions that do not directly contact the inner ears E. maculatus. This may explain why it does not reveal linear threshold shifts similar to other hearing specialized fish. Ramcharitar and Popper (2004) These observations in sciaenids contradict previous studies and the current data in cichlids. It is unexpected that two species exhibiting similar baseline thresholds are similarly affected by a certain noise level (124 dB) and differently affected by a 12 dB higher noise level. In the goldfish Lepomis gibbosus, which had lower auditory sensitivities compared to both otophysines, with maximum sensitivity of 98 dB at 100 Hz, white noise of 110 dB did not affect its hearing sensitivity at any frequency. When sunfish were exposed to 130 dB noise level, the thresholds shifted up to 11 dB (500 Hz) compared to baseline thresholds.Our intra-familial results in cichlids are in good agreement with masking studies that compared species specialized for hearing, such as otophysines, and species not specialized, such as centrarchids and percids Cyprinus carpio and the European perch Perca fluviatilis. The former is an otophysine and the latter is a non-specialized percid. Again, the same noise types resulted in quite different masking effects. In the carp, hearing sensitivity (highest sensitivity: ∼60 dB) declined at all types of habitat noise but particularly at the highest level by up to 49 dB. In contrast, the perch was less sensitive than the carp (baseline hearing thresholds 20 dB higher than that of the common carp). The presence of different masking noise types had slight (0–7 dB) or moderate effects on the hearing thresholds. Hearing thresholds were maximally elevated by 12 dB (200 Hz) in the case of the river noise.Amoser and Ladich (2005) The above intra-familial and inter-familial comparative studies focused on the effects of noise on different hearing abilities. Several other studies analyzed the degree of masking under natural ambient noise conditions. The general observation was that masking effects (threshold shifts as compared to baseline thresholds) were small under quiet conditions such as in the absence of wind, rain or surf. Major threshold shifts were found under noisy conditions in fish possessing higher hearing abilities.Gadus morhua and other representatives of the family Gadidae in a Scottish Loch 15 m below the sea surface and 5 m above the sea bed. Unmasked thresholds were obtained only at calm sea conditions. More recent studies on non-related marine fish taxa such as batrachoidids, sciaenids, pomacentrids and gobiids revealed that the hearing sensitivities were only slightly masked. Vasconcelos et al. (2007) Halobatrachus didactylus compared to quiet lab conditions. Codarin et al. (2009) Gobius cruentatus, the brown meagre Sciaena umbra (family Sciaenidae) and the Mediterranean damselfish Chromis chromis (Pomacentridae) changed by less than 3 dB when exposed to the ambient noise recorded in their habitat, the Miramare Natural Marine Reserve in the Adriatic Sea. Observations in freshwater fishes revealed similar trends. Amoser and Ladich (2005) Pseudorasbora parva, a common Eurasian cyprinid, has best hearing sensitivities between 300 and 800 Hz (57 dB re 1 µPa) under quiet lab conditions. In the presence of ambient noise of its natural habitat (Lake Neusiedl), best thresholds shifted maximally 15 dB Chapman and Hawkins (1973) S. tinanti or in the round goby Neogobius melanostomusWhy did fish evolve enhanced hearing abilities when their hearing may be masked in their natural habitats? Accessory hearing structures and thus enhanced sensitivities may have evolved to enable fish in calm habitats to detect sound of different origin such as from conspecifics , heterospecifics  or abiotic sources . Detecting and intercepting these sound sources at larger distances either permanently or during calm periods or in quiet regions of their distribution range may have been a major selective advantage over less specialized species. On the other hand, the presence of large swim bladders can be disadvantageous for bottom-dwelling fish due to buoyancy, particularly in running waters cf. . If thesIn summary, the present study demonstrates that hearing sensitivities and subsequently masking can differ considerably within members of one bony fish family. This indicates that they live under different eco-acoustical conditions, i.e. different ambient noise regimes. Based on masking studies, we suggested that eco-acoustical constraints are probably the main selective forces shaping hearing sensitivities in fishes.


This case illustrates that superior semicircular canal dehiscence syndrome can be associated with a "pseudo"-conductive hearing loss, a symptom that overlaps with the clinical appearance of otosclerosis.We present the case of a 48-year-old German Caucasian woman presenting with hearing loss on the left side and vertigo. She had undergone three previous stapedectomies for hearing improvement. Reformatted high-resolution computed tomographic scanning and the patient's history confirmed the diagnosis of concurrent canal dehiscence syndrome.Failure of hearing improvement after otosclerosis surgery may indicate an alternative underlying diagnosis which should be explored by further appropriate evaluation. Superior semicircular canal dehiscence is an abnormal exposure of the vestibular membranous labyrinth in the middle cranial fossa. Superior semicircular canal dehiscence syndrome (SCD) occurs when a loss of the bone normally covering the superior semicircular canal in the middle cranial fossa produces one or more of the following symptoms: conductive hearing loss, acute pressure- and sound-evoked vestibular symptoms and chronic dysequilibrium . The corWe present the case of a 48-year-old German Caucasian woman who presented with hearing loss on the left side and vertigo. The patient had a history of three previous stapedectomy operations carried out elsewhere to improve her hearing loss Figure . The firAfter the third operation, the patient came to our unit with persistent amblyacousia as well as severe vertigo and headache. Pure tone audiometry showed a maximal conductive hearing loss. The patient located in her left ear the sound of a tuning fork pressed on the right ankle. This phenomenon suggested SCD. Further high-resolution computed tomographic (CT) scans and audiometery were performed. A CT scan revealed superior semicircular canal dehiscence Figure .We hypothesize that the otosclerotic focus in the oval window prevented the development of symptoms from this patient's SCD. Surgical stapedectomy created a third window and resulted in immediate postoperative imbalance and auditory symptoms.SCD is one of the best documented and most investigated third-window lesions of the inner ear. We posit that patients with persistent audiovestibular symptoms after stapes surgery should be examined for the presence of SCD .A combination of high-resolution CT scans and audiometry is recommended for diagnosis. The audiometric signs of SCD are conductive hearing loss with low-frequency bone conduction threshold better than 0dB(HL) and normal tympanometry with intact acoustic reflexes.Auditory manifestations include hyperacusis to bone-conducted sounds and conductive hearing loss with normal acoustic reflexes. A directed patient history, documentation of upward and torsional nystagmus evoked by sound and/or pressure and radiologic findings are helpful in the diagnosis of SCD.Acoustic reflexes and vestibular evoked myogenic potentials (VEMPs) aid in the identification of patients with an apparent conductive hearing loss with normal acoustic reflexes or those patients who are found to have an asymptomatic dehiscence on radiology . The treThe typical audiometric findings are of an air-bone gap in the low and middle frequencies  with no gap or only a small gap at higher frequencies. The low-frequency  bone conduction thresholds are sometimes at supranormal levels, 0 to -20 dB or better. The lack of middle ear pathologic findings as a cause of the conductive hearing loss (CHL) in SCD has been well documented by a variety of diagnostic tests, such as tympanometry, acoustic reflexes, laser Doppler vibrometry, air-conducted VEMP testing, otoacoustic emission testing and exploration of the middle ear -13. DefiIn choosing treatment options, the severity of symptoms in each individual patient should be considered. Patients with minimal or minor symptoms should avoid provocative stimuli and undergo supportive measures such as vestibular rehabilitation or vestibular suppressants. These patients may require longer follow-up to ensure symptom resolution. Patients with disabling sound- or pressure-induced vertigo, imbalance or oscillopsia may require surgical treatment. The standard surgical options include middle fossa craniotomy for superior canal occlusion or resurfacing and transmastoid superior semicircular canal occlusion . The aimThe authors declare that they have no competing interests.Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.ML and JE analyzed and interpreted the patient data regarding the otorhinolaryngological disease. TU and HS were major contributors in writing the manuscript. All authors read and approved the final manuscript.


IIIXO) formation was established. This method showed that due to the conversion of FeII to FeIII by H2O2 and subsequent formation of FeIIIXO complex halo in agar medium, the logarithm of H2O2 concentration from 5 to 160 µM was linearly correlated to the diameter of purplish red FeIIIXO halo. By extracting the LAAO-generated H2O2 concentration, the LAAO activity can be quantitatively determined. This FeIIIXO agar assay is highly sensitive to detect H2O2 down to micromolar range. More importantly, it is easy to handle, cheap, reproducible, convenient and accurate. Coupled with SDS-PAGE, it can directly be used to determine the number and approximate molecular weight of LAAO in one assay. All these features make this in-gel FeIIIXO assay useful and convenient as a general procedure for following enzyme purification, assaying fractions from a column, or observing changes in activity resulting from enzyme modifications, hence endowing this method with broad applications.L-amino acid oxidase (LAAO) has important biological roles in many organisms, thus attracting great attention from researchers to establish its detection methods. In this study, a new quantitative in-gel determination of LAAO activity based on ferric-xylenol orange (Fe The quantitative detection of H2O2 is mostly done by measuring the chemiluminescence due to the addition of horseradish peroxidase (HRP) and its substrate. There exists the commercially available kit , with detection limit down to micromolar level. 2O2 produced by LAAO activity acts as electron donor. Although the Prussian blue agar assay is cheaper and more convenient than the HRP-based assays, there are still several drawbacks that may limit its further application. Firstly, its quantitative detection limit is only down to about 0.5 mM level of H2O2. Secondly, potassium hexacyanoferrate (III) itself is safe, but under the peracidic condition it may degrade and release extremely toxic CN−. Thirdly, Prussian blue is a complicate class of chemical compounds containing Prussian blue, Prussian brown, Prussian white and Berlin green, which is sensitive to pH condition. Therefore, extremely careful pH adjustment in the Prussian agar preparation is required to produce reproducible result of color formation. It is desirable to develop an assay combining the advantages of HRP-based and Prussian blue-based measurement.L-amino acid oxidases  function in catalyzing the transformation of L-amino acids to the corresponding a-keto acids with the release of ammonium and hydrogen peroxide (HII) can be oxidized to ferric ion (FeIII) in the presence of H2O2, previous studies have demonstrated that XO can be applied to measure H2O2 by spectrophotometrically analyzing the purplish red complex  that is formed by FeIII and XO 2O22O2 sensitivity, compared to Prussian blue agar assay. The purpose of this study is to describe a new application of the FeIIIXO formation for quantitatively determining the LAAO activity by in-gel visualization and measurement of H2O2. This new FeIIIXO assay is not only comparative to the HRP-involved assay in terms of sensitivity, but also bears similar benefits of Prussian blue agar assay, including ease of handling and cost-effectiveness. Moreover, it can be directly used for in-gel determination of the number and molecular weight of LAAO on the SDS-PAGE after visualization of the purplish red FeIIIXO complex.Like Prussian blue, xylenol orange  aminomethyl]-o-cresolsulfonephthalein) is one of the most important color materials. Considering that ferrous ion , the proper concentration of H2SO4 for FeIIIXO agar assay is 6 mM up to 10 mM, giving final pH of 3.5 down to 2.3. After pouring, the FeIIXO agar plates consistently gave shallow orange red color as XO (pH indicator) itself will show orange red under acidic condition.The formation of Fe µM H2O2 . No purp4 and XO are the two major elements in FeIIXO agar. To investigate the effect of the molar ratio of FeSO4 to XO on the FeIIIXO agar assay, FeSO4 with different concentrations from 0 to 0.4 mM were added to assay medium while XO was fixed at 0.15 mM. As shown in 2O2 condition, the diameter of purplish red halo increased with higher concentration of FeSO4 and appeared to a maximum when the FeSO4 concentration reached 0.25 mM. Further increase of FeSO4 concentrations from 0.25 mM to 0.4 mM cannot obviously enlarge the purplish red zone, indicating that the FeIIIXO was saturated when the molar ratio of FeSO4 to XO reached 5∶3.FeSOIIXO agar medium was pH-sensitive, we also investigated the effect of pH on the color development of FeIIIXO agar. The background solutions with different pH values ranging from 1 to 14 were prepared by mixing 6N HCl with 6N NaOH as required. As indicated in row 1 of IIIXO agar, remaining the original orange red. However, both lower pH and higher pH did make the color change of the agar medium. The background solutions with pH≤2 yielded lemon yellow halos, most likely due to the color presentation of XO as a pH indicator under peracidic condition. The lower the pH, the bigger and stronger the lemon yellow halos. On the other hand, when the background pH was above 12, the purplish red halos were generated even without H2O2 treatment  with the same pH of 7.5 also gave uniform and reproducible purplish red halos caused by the released H2O2  with the increase of H2O2 concentration from 0 to 160 µM, but not significant (P>0.05) with the further increase of H2O2 concentration from 160 µM to 250 µM , where x is the diameter and y the H2O2 concentration. Further plotting in 2O2 concentration in a range of 5 µM to 160 µM with linear fit under an equation y = 2.049×−1.460 (R2 = 0.997), where x is the diameter and y the logarithm of H2O2 concentration.To quantify the LAAO activity, we first prepared a series of standard H2O2 concentration fashion using the above extracted equation, R3-LAAO was used to oxidize the substrates L-Leu and L-Met, respectively, in separate reactions. After oxidization, the reaction solution was diluted 50 times and then subjected to FeIIIXO agar assay. The results in 2O2 concentrations of 83.3 µM and 41.7 µM, respectively, on the basis of the above extracted equation in 2O2 with concentrations of 83.3 µM and 41.7 µM were applied to FeIIIXO agar assay and finally yielded the purplish red halos with diameters of 1.65 cm and 1.50 cm, respectively, all agreeing with our calculated concentrations. Therefore, the R3-LAAO activities with L-Leu and L-Met as substrates were 0.833 U/mL and 0.417 U/mL, respectively. All these findings indicate that the FeIIIXO agar assay is feasible to sensitively detect the H2O2 produced by LAAO activity and the extracted equation is reliable to quantitatively determine the LAAO activity. To verify this method, another enzyme source, the commercial Crotalus adamanteus venom LAAO (caLAAO) was also used to oxidize L-Leu and applied to FeIIIXO agar assay after 400 times dilution. 2O2 based on the extracted equation. When 17.3 µM standard H2O2 was applied to FeIIIXO agar assay, a purplish red zone with 1.30 cm diameter appeared. To further confirm its reliability, the oxidization solution of L-Leu by caLAAO was serially diluted by 100 times, 200 times, 300 times and 400 times, and subsequently subjected to FeIIIXO agar assay. The results  with different concentrations of 63.3 µM, 34.5 µM, 23.0 µM and 17.5 µM gave purplish red halos with diameters of 1.60 cm, 1.46 cm, 1.38 cm and 1.30 cm, respectively, all agreeing with our calculated results. All these findings indicate that FeIIIXO agar assay is very reliable for quantitatively detecting the LAAO activity.To evaluate the LAAO activity determined as H results  showed t2O2 concentration is around 500 µM IIIXO agar assay is much more sensitive (around 100 times higher). To further compare their sensitivity, R3-LAAO was used to oxidize L-Cys, L-Glu, L-Asp, L-Val, L-Ala, L-Ser, L-Gly and L-Pro, in separate reactions, and subsequently the generated H2O2 was measured with both Prussian blue agar assays and FeIIIXO agar assay. Results showed that no clear color was developed in Prussian blue agar plate , indicating that R3-LAAO in SDS-PAGE bears only one active unit. Similarly, the two sample replicates (lanes 3 and 4) with CBB staining also gave a clear purplish red band at the same migration position on FeIIXO agar, revealing that R3-LAAO used in this assay is resistant to SDS and β-mercaptoethanol. As expected, the duplicate lanes 3 and 4 additionally showed CBB-stained protein bands in SDS-PAGE since R3-LAAO sample was precipitated from fermentation crude of Psudoalteromonas sp. R3 IIXO agar were sliced out from a CBB-stained lane-4 replicate lane, and put on FeIIXO agar (lane 5). It was found that even after long time exposure to multiple steps treatment, the sliced target protein band with LAAO activity in SDS-PAGE still clearly yielded a purplish red band on FeIIXO agar, indicating that LAAO is very stable in this SDS-PAGE coupled FeIIIXO agar assay. According to the molecular weights of CBB-stained standard protein marker (lane M), the corresponding protein band with LAAO activity from Psudoalteromonas sp. R3 was estimated to be around 65 kDa in size on SDS-PAGE, which is in agreement with the one achieved by SDS-PAGE coupled Prussian blue agar assay Different methods have been developed to determine the LAAO number and its molecular weight IIIXO complex driven by H2O2 is a linear function of logarithm of H2O2 concentration from 5 µM to 160 µM, thus allowing this assay to quantitatively determine the LAAO activity with a similar sensitivity as the HRP-involved methods. Although HRP is H2O2 sensitive probe, the assay is complex and expensive. Moreover, the solution of HRP is unstable and needs to be used right after it is ready. Besides, it needs detection instrument. In contrast, FeIIIXO agar assay does not rely on any detection instrument, and it is simple, stable and cost-effective. Compared with Prussian blue agar assay IIIXO agar assay is more environmentally friendly. More importantly, it gives two orders of magnitude improvement in sensitivity (5 µM vs. 0.5 mM). Considering its high sensitivity and convenience, this FeIIIXO agar assay can be used to differentiate the mutants with slight difference in LAAO activity from a mutant library with altered expression of LAAO, saving a great number of workload for the investigation of the involved regulation mechanisms underlying the LAAO production.The diameters of the purplish red halos of FeIIXO agar before assay. Under non-acidic condition, the entire FeIIXO agar medium will soon become purplish red even in the absence of H2O2 (data not shown). Most probably, FeII in medium is unstable and will be easily oxidized by oxygen to FeIII which will sequentially coordinate with XO to form purplish red FeIIIXO complex. During the fabrication of FeIIXO agar, H2SO4 should be added to medium before FeSO4. Otherwise, the whole mixture of assay medium will also immediately turn to purplish red (data not shown). Another reason for acidic condition in FeIIXO agar medium is that it can help to avoid the hydrolysis of the iron IIXO agar, the acidic capacity or low pH in agar medium is also required for the formation of FeIIIXO complex driven by H2O2. As shown in IIXO agar treated with 40 µM H2O2 if if FeIIXO agar is lack of H2SO4 to give final pH 6. However, extreme acidic condition (pH 1.8 in the presence of 13 mM H2SO4) will make the FIIXO agar medium become lemon yellow, and obscure the color development of FeIIIXO complex. It is most likely that XO is responsible for the lemon yellow color of the agar medium under pH 1.8. Based on the above observation, a final pH value between 2.3 and 3.5 of the agar medium by supplying 6 mM to 10 mM H2SO4 is recommended for proper color development of the assay. We also show that the pH of the detection solution is crucial to the success of FeIIIXO agar assay. Proper color development of FeIIIXO agar is only observed when the detection solution has a pH between 4 and 11. When the pH is ≤2, XO itself will give strong lemon orange and thus mask the color development of purplish red FeIIIXO complex caused by H2O2. When the pH is ≥12, there are two more sources responsible for the purplish red color development in addition to FeIIIXO complex formation due to H2O2. First, XO itself will show purplish red color at this pH; second, under peralkaline condition, other oxidants, such as oxygen, can also easily convert FeII to FeIII, which will subsequently react with XO to form purplish red FeIIIXO. Therefore, the detection of purplish red FeIIIXO formed by H2O2 is not possible at pH above 12. Fortunately, the fermentation solutions from LAAO-producing microorganisms or direct LAAO enzymatic reaction solutions usually have pH values in the range of 4 to 11, thus giving this method broad applicability.As reported IIIXO has a 1∶1 stoichiometry IIIXO halo reaches equilibrium when the molar ratio of FeSO4 to XO in medium is close to 2∶1. XO and FeIII can form FeIII2XO, FeIIIXO and FeIIIXO2 since XO is a bi-functional metallochromic reagent, mainly depending on the molar ratio of iron ion to XO IIIXO2) will form if XO is in excess. When the molar ratio of iron to XO approaches to 1∶1, the 1∶1 complex (FeIIIXO) becomes predominant. In contrast, FeIII2XO will predominate in the presence of excess iron, which is attributed to XO's two isolated iminodiacetate groups that can bind metal ions. This is in agreement with our observation.It has been reported that the complex FeIIIXO agar assay can be directly used to determine the numbers and approximate molecular weights of LAAO protein in one assay, giving crucial advantages over conventional spectrophotometric or fluorometric measurement. Without heating, the LAAO used in this study can tolerate SDS and β-mercaptoethanol, and maintain its activity even after long time exposure to the CBB-staining procedure and de-staining solution with glacial acetic acid. It is clear that knowing exactly the numbers and molecular weights of LAAO can benefit further purification and characterization of this enzyme. In particular, the sliced target band with LAAO activity can directly be analyzed with different techniques, such as protein sequencing and LC-MS/MS analysis.Coupled with SDS-PAGE, FeIIIXO agar assay can be performed based on the visual measurement rather than the spectrophotometric or fluorometric colorimetry, thus providing it with broad advantages of simplicity and cost-effectiveness. To push the visual threshold detection for trace H2O2 caused by LAAO activity, adjustment of the acidic condition of FeIIXO agar and near 2∶1 molar ratio of FeSO4 to XO are highly necessary. Besides, the addition of D-sorbitol to FeIIXO agar medium can also increase the sensitivity of this FeIIIXO agar assay With agar in medium, our Fe2O2 was purchased from Shanghai Lingfeng Chemical Reagent CO., LTD (China). Xylenol orange  and ferrous sulfate were purchased from SANGON BIOTECH , and D-sorbitol supplied by Biosharp CO., LTD (China).All chemicals are of at least analytical grade and used without further purification. HPsudoalteromonas sp. R3 (R3-LAAO) was harvested as reported Crotalus adamanteus venom (caLAAO) was purchased from Worthington Biochemical Corporation, USA.LAAO from the marine bacterial IIIXO agar assay was performed as follows: (1) prepare solution of ferrous-XO (FeIIXO) with 0.25 mM FeSO4, 6 mM H2SO4, 0.15 mM XO, 0.1 mM D-sorbitol and 1.5% agar; (2) dissolve the mixture completely at 100°C for 5 min and pour into glass Petri dish to make agar plate; (3) make circular wells on agar plate with a hole puncher whose diameter is 6 mm; (4) add 50 µL detection solutions containing standard H2O2 or H2O2 produced by LAAO activity to each well and wait for 60 min at room temperature for color change; (5) visualize the FeIIIXO formation and measure the size of purplish red halo.Unless otherwise stated, FePsudoalteromonas sp. R3 culture supernatant IIIXO agar assay with appropriate dilution or Prussian blue agar assay without dilution The stereospecific oxidation reaction was performed with 10 mM of each amino acid in 10 mL of R3-LAAO solution harvested from 2O2 solutions with different concentrations ranging from 0.5 µM to 250 µM with uniform pH 7.5 were subjected to FeIIIXO agar assay; (2) after assay, the diameters of the formed purplish red FeIIIXO halos were measured and the correlation equations between H2O2 concentrations and purplish red halo diameters were extracted using Origin software; (3) 50 µL oxidization solutions of L-amino acid by LAAO were applied to FeIIIXO agar assay and the diameters of the generated purplish red halo were recorded; (4) the concentration of H2O2 produced by LAAO activity was calculated based on the extracted correlation equations between H2O2 concentrations and purplish red halo diameters; (5) the LAAO activity was determined with the fashion of the produced H2O2 concentration. One unit (U) is defined as the amount of enzyme that catalyses the formation of 1 mM H2O2/h at 37°C.Unless otherwise stated, the determination of the LAAO activity includes the following steps: (1) 50 µL standard HIIIXO agar assay consists of the following three steps: (1) SDS-PAGE electrophoresis. The several replicates of detection samples containing LAAO were mixed with 4-fold sample loading buffer . Without heating, 20 µL resultant mixtures were separately applied to each well of normal SDS-PAGE with 5% stacking gel and 12% separation gel, as described by Laemmli IIIXO agar assay using LAAO-contained gel. After SDS-PAGE electrophoresis, the entire gel was washed once with distilled water and cut into two pieces. One piece was directly put on FeIIXO agar for the color change; the other with the sample replicates was first stained with Coomassie brilliant blue (CBB) IIXO agar aside for the color development after three times wash with distilled water; (3) targeting of the protein with LAAO activity. After visualization of purplish red band on FeIIXO agar (below SDS-PAGE), the protein bands directly above the formed purplish red band area were cut out from the sample replicate of CBB stained SDS-PAGE and put on FeIIXO agar to determine the target band with LAAO activity which caused the formation of purplish red. If necessary, the standard protein ladder was used for the determination of molecular weight of target LAAO.Unless otherwise described, SDS-PAGE coupled FeTable S12O2 with different concentrations by ANOVA.Statistical analysis of dependent variable diameters of purplish red halos from H(DOC)Click here for additional data file.Figure S12SO4 with different concentrations in FeIIXO agar medium on the formation of purplish red FeIIIXO complex caused by 40 µM H2O2.Effect of acidic condition derived from H(TIF)Click here for additional data file.Figure S24 in FeIIXO agar medium on the formation of purplish red FeIIIXO caused by 40 µM H2O2.Effect of FeSO Xylenol orange (XO) was fixed at 0.15 mM.(TIF)Click here for additional data file.Figure S3IIIXO agar assay for determination of Reliability of FeCrotalus adamanteus LAAO (caLAAO) activity. The oxidization solutions of L-Leu by caLAAO were serially diluted by 100 times, 200 times, 300 times and 400 times, and 50 µL diluted solutions were subjected to FeIIIXO agar assay (left hole). On the basis of the diameters of the formed purplish red FeIIIXO halos, the concentrations of H2O2 produced by LAAO activities were calculated with the equations in 2O2 solutions (right hole) as indicated above the corresponding halos were used to confirm the accuracy of FeIIIXO agar assay. The diameters of the purplish red halos were marked below the holes.(TIF)Click here for additional data file.Figure S4IIIXO agar assay for the characterization of LAAO from SDS-PAGE coupled in-gel FePseudoalteromonas sp. R3 (R3-LAAO). After electrophoresis, different lanes of SDS-PAGE with replicated samples were sliced out for different treatments and subsequently put together on FeIIXO agar for color development. Lane M: standard protein marker stained with Coomassie brilliant blue (CBB); Lanes 1 and 2: duplicate LAAO samples from Pseudoalteromonas sp. R3 (R3-LAAO) IIXO agar (below SDS-PAGE) and its molecular weight was around 65 kDa.(TIF)Click here for additional data file.


Blastocystis sp. is currently the most common intestinal protist found in human feces and considered an emerging parasite with a worldwide distribution. Because of its potential impact in public health, we reinforced the picture of Blastocystis sp. prevalence and molecular subtype distribution in Africa by performing the first survey of this parasite in Senegal.Blastocystis sp. by non-quantitative and quantitative PCR using primer pairs targeting the SSU rDNA gene. Positive samples were subtyped to investigate the frequency of Blastocystis sp. subtypes in our cohort and the distribution of subtypes in the symptomatic and asymptomatic groups of children.Stool samples from 93 symptomatic presenting with various gastrointestinal disorders or asymptomatic children living in three villages of the Senegal River Basin were tested for the presence of Blastocystis sp. indicating a striking parasite prevalence of 100%. Mixed infections by two or three subtypes were identified in eight individuals. Among a total of 103 subtyped isolates, subtype 3 was most abundant (49.5%) followed by subtype 1 (28.2%), subtype 2 (20.4%) and subtype 4 (1.9%). Subtype 3 was dominant in the symptomatic group while subtypes 1 and 2 were detected with equal frequency in both symptomatic and asymptomatic groups. The distribution of subtypes was compared with those available in other African countries and worldwide. Comparison confirmed that subtype 4 is much less frequently detected or absent in Africa while it is commonly found in Europe. Potential sources of Blastocystis sp. infection including human-to-human, zoonotic, and waterborne transmissions were also discussed.By the use of molecular tools, all 93 samples were found to be positive for Blastocystis sp. in our Senegalese population was the highest prevalence ever recovered worldwide for this parasite by reaching 100%. All cases were caused by subtypes 1, 2, 3 and 4 with a predominance of subtype 3. More than half of the children infected by Blastocystis sp. presented various gastrointestinal disorders. Such high prevalence of blastocystosis in developing countries makes its control a real challenge for public health authorities.The prevalence of  Blastocystis sp. is an anaerobic protist that inhabits the gastrointestinal tract of humans and many groups of animals[Blastocystis sp.[Blastocystis sp. Moreover, a higher risk of infection has been identified in food and animal handlers, providing conclusive evidence on the transmission of the parasite between humans and animals[ animals-4. This  animals is often animals. Indeed, animals,7 and re animals,9. These animals-12. Such animals, waterbostis sp.. Numeroustis sp. highligh animals,17.Blastocystis sp. isolates from humans and animals have been reported to be roughly indistinguishable[Blastocystis sp. isolates has been reported based on the comparison of the small-subunit (SSU) rDNA gene sequence[Blastocystis sp. isolates into subtypes (STs)[Blastocystis sp. is attributable to ST3 but the distribution of the four predominant STs may vary among the areas studied. Thereby, ST4 is almost as common as ST1 and ST3 in some European countries but seems rare or completely absent in Africa, Asia, and America[At the morphological level, uishable. Howeversequence leading es (STs). To datees (STs) but it ies (STs). Indeed, America,20. The Blastocystis sp. can be found in both symptomatic and asymptomatic patients its clinical relevance remained uncertain[in vivo, in vitro and genomic data strongly suggests that this microorganism may be pathogen[Blastocystis sp. should be associated with a variety of non-specific gastrointestinal disorders including diarrhea, abdominal pain, nausea and vomiting[Blastocystis sp. has increasingly been implicated in diarrheal illness in immunocompromised individuals including HIV/AIDS and cancer patients and transplant recipients[As ncertain. Howeverpathogen-24 and apathogen particulpathogen,27. Thervomiting,22 and avomiting,28 and cvomiting,30. In acipients,32.Blastocystis sp. may be dependent upon ST but recent epidemiological data remain contradictory[Blastocystis sp. coupled to its high prevalence in the human population raise crucial questions about its current burden in public health. Consequently, information on the prevalence of the parasite and the distribution of STs are starting to emerge in still poorly studied geographic areas as Africa. To date the available data concern only few African countries of the North and West such as Egypt[Blastocystis sp. prevalence and ST distribution in Africa by performing the first survey of this parasite in Senegal. In this epidemiological study, we described the prevalence of Blastocystis sp. infection and ST distribution in a cohort of Senegalese children living in 3 rural villages in close contact with animals and with poor or no access to clean water and sanitation. The distribution of Blastocystis sp. STs in both asymptomatic and symptomatic individuals was investigated together with potential risk factors for transmission of the parasite.It has long been suggested that the pathogenesis of adictory,29,33-36as Egypt,37,38, Las Egypt and one as Egypt. Therefohttp://www.espoir-sante.org). The present study was approved by the National Ethics Committee of the Ministry of Health of Senegal . Oral and written informed consents were obtained from the parents or the legal guardians of the children in accordance with the Code of Ethics of the World Medical association (Declaration of Helsinki). Children enrolled in the present study formed a sub-cohort of the "AnoPalAnoVac" project  and were recruited on the basis of their age (6 to 10 years of age in October 2011) through village nurses, healthcare workers in the community and school directors. Date of birth was ascertained from vaccination cards or school register. A standardized questionnaire was completed for each child , sponsored by the Biomedical Research Centre EPLS  . For each DNA sample, the non-qPCR product with the highest intensity on agarose gel was purified and sequenced. Direct sequencing of few non-qPCR products generated mixed signals that could reflect infections by different STs or was of poor quality probably due to a too small amount of parasite DNA matrix. Because of the difficulty in interpreting the corresponding DNA sequencing chromatograms, same samples were re-analyzed by non-qPCRs and positive bands of the expected sizes were purified and cloned as previously described[Blastocystis sp.-negative stool) subsequently used in qPCR assays and negative (DNA matrix replaced by water) and positive (DNA obtained from a Blastocystis sp. ST4 culture) qPCR controls. The expected 320 bp-amplified variable region of the SSU rRNA gene was directly sequenced for subtyping. The SSU rRNA gene sequences obtained in this study have been deposited in GenBank under accession numbers KF848509 to KF848624. Obtained sequences were compared with all Blastocystis sp. SSU rRNA gene sequences available from the National Centre for Biotechnology Information (NCBI) using the BLAST program. STs were identified by determining the exact match or closest similarity against all known Blastocystis sp. STs according to the updated classification by Alfellani et al.[Samples stored in S.T.A.R. buffer were stirred and then centrifuged for 1 min at 1,000 g. Total genomic DNA was extracted from 200 μl of the cleared stool supernatant using the QiaAMP DNA Stool Mini Kit  according to the manufacturer’s instructions. For each sample, 5 μl of extracted DNA was submitted to non-quantitative Polymerase Chain Reactions (non-qPCRs) as previously described using two independent pairs of a et al. and Poirisolates. Non-qPCescribed. Three pBlastocystis sp. by non-qPCR and qPCR methods using specific primer pairs amplifying distinct domains of the SSU rDNA coding region[Blastocystis sp. by non-qPCR methods. The remaining 22 samples negative by non-qPCR nevertheless proved to be positive for the parasite by qPCR. Thereby, by means of these different molecular approaches, Blastocystis sp. was recognized in 100% of stool samples of this Senegalese population. To our knowledge this represents the highest prevalence ever recovered worldwide for this parasite.In this study, fecal samples were collected from 93 children, 46 boys and 47 girls, aged 6 to 10 years and living in 3 villages situated in the north of Senegal. This cohort consisted of two groups: one symptomatic composed of 54 children presenting variously with abdominal pain, vomiting, and diarrhea and one asymptomatic including 39 other children  primers or genus-specific primers with subsequent sequencing for ST identification. Several regions in the SSU rDNA gene have been proposed by different authors for sequencing[Blastocystis sp. diagnosis and subtyping[Blastocystis sp. ST in the same sample. Therefore subtyping of isolates in mixed infections requires cloning of the non-qPCR product and sequencing of several clones as previously suggested[Blastocystis sp. by non-qPCR methods in our study. Hence, at this stage of our screening, the prevalence of the parasite was 76%, which greatly underestimated its true prevalence as determined in a second step by qPCR (100%). Consequently, our study confirmed the impact of detection methods to ascertain the actual prevalence of Blastocystis sp. in human populations and the interest and higher sensitivity of qPCR assays in comparison to non-qPCR methods for epidemiological surveys.In almost all molecular epidemiological studies conducted to date, only one nucleic acid-based diagnostics methodology was used to identify and subtype quencing but the quencing has beenquencing,42 and aubtyping. Howeveruggested. In addiBlastocystis sp. never reached 100% in any cohort studied so far, it was described as very high in some developing countries specially, in children. By using non-molecular standard methods, the prevalence of the parasite already exceeded 40% in communities in Venezuela[Blastocystis sp. was around 60% in immigrants in the city of Naples  who came for a large majority from northern and western African countries[Blastocystis sp. had never been determined before, intestinal parasites (protozoa and helminths) represent for a long time a major public health problem. Indeed, the global prevalence of intestinal parasites in children ranged from 32% to 84% depending on the study areas[Blastocystis sp. in the current study.Although the prevalence of enezuela, Peru[4enezuela, Colombienezuela, and in enezuela, Thailanenezuela, and Lebountries. In Libyountries. In Senedy areas-53. SuchBlastocystis sp. isolates reported so far. The comparison with representative sequences of all known Blastocystis sp. STs allowed the direct subtyping of the new isolates  or three STs (2 samples) were identified  followed by ST1 (28.2%), ST2 (20.4%) and ST4 (1.9%). The distribution of STs was almost similar between the three villages with a majority of infections attributable to ST3. The only significant difference was that ST1 followed at position 2 in Niandiane and Agniam Towguel and at position 3 in Fanaye Diery.In all the children, the expected SSU rDNA domains targeted by non-qPCR and qPCR methods were amplified and successfully sequenced. Each of the new SSU rDNA gene sequences obtained in this study showed very high similarity 98% to 100%) to homologous sequences of the other 00% to hohree STs  samples Blastocystis sp. STs in Senegal is quite similar to that observed in a majority of countries all over the world with a predominance of ST3 followed by ST1 then ST2 or ST4[The distribution of 2 or ST4. If we f2 or ST4,37,38, L2 or ST4, the lat2 or ST4, it can 2 or ST4,34. Howe2 or ST4,38. Inde2 or ST4 and rareThe prevalence of mixed ST infections was 8.6% (8/93 samples) in our Senegalese cohort. This percentage is probably underestimated but roughly similar to that calculated (6%) by taking into account subtyped isolates from all epidemiological studies published to date (191 of 3171 samples) and listed by Alfellani et al.. Mixed SBlastocystis sp. infection , the ST distribution was evaluated in our cohort of 93 symptomatic or asymptomatic children. Interestingly, the majority of children infected by Blastocystis sp. (54/93 or 58%) presented various gastrointestinal disorders as previously observed in a pediatric unit in Turkey[According to recent epidemiological reports, links between ST and pathogenicity are still matter of debate,33-36,41n Turkey. In the n Turkey,58, and n Turkey. Regardin Turkey, Turkeyn Turkey, Iran[3n Turkey, Egypt[n Turkey, and Lebn Turkey,61 and wn Turkey. In contn Turkey suggesten Turkey. GloballBlastocystis sp. like the other common gastrointestinal parasites[Blastocystis sp. Indeed, recent studies have revealed a human-to-human transmission coupled with a high prevalence of the parasite in a close community of girls in Thailand[Blastocystis sp. would be another significant source of infection due to the wide range of animals potentially infected with the parasite and in close contact with humans[Blastocystis sp. although large-scale waterborne outbreaks involving the parasite have not yet been documented. Ability of Blastocystis sp. cysts, the only transmissible stage of this parasite to survive for long periods of time in the environment together with their low size[Blastocystis sp. has also been added by the World Health Organization to the list of waterborne parasites[Blastocystis sp. was previously reported in drinking water, rivers, tanks, and sewage samples[Blastocystis sp. and should be considered as serious potential threats to consumers. Further, the analysis of fecal samples from animals living in or near the Senegalese villages and of water samples would help to confirm their epidemiological importance as sources of infection and contamination for inhabitants.The fecal-oral route is the main mode of transmission of arasites. To reacThailand as well Thailand whose deh humans,4,55,56.low size,15 reprearasites. Blastoc samples,14,15,64Blastocystis sp. in Senegal that, moreover, greatly expands the few data available on molecular epidemiology of this parasite in Africa. In this country, the prevalence of Blastocystis sp. in a cohort of children living in a rural area reached a peak of 100% never achieved before. Such high prevalence may reflect different exposure of individuals to animal (zoonosis) and environmental (waterborne) sources of infection together with large-scale human-to-human transmission. This study also raises questions about the real pathogenicity of Blastocystis sp. since more than half of children infected by this parasite presented various gastrointestinal symptoms and highlights the socioeconomic impact of blastocystosis in developing countries with low environmental conditions and quality of life. Finally, this epidemiological survey provides necessary information to public health authorities for urgently setting prevention  and control programs that should help to reduce the burden of Blastocystis sp.To our knowledge, this is the first investigation of prevalence and ST distribution of ST: Subtype; IBS: Irritable bowel syndrome.The authors declare that they have no competing interests.LG and MS collected stools and patient data and helped in drafting the manuscript. DES, DM, AC, PP and IW collected and analyzed PCR and sequence data and helped in drafting the manuscript. EV, GR, MH, LD, FDe and FDa conceived, designed and coordinated the study and drafted the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/14/164/prepubStandardized questionnaire completed for each child in the study.Click here for file