Cherax quadricarinatus (designated Cq-IAG), was found in this study to be the prominent transcript in an AG cDNA subtractive library. In C. quadricarinatus, sexual plasticity is exhibited by intersex individuals in the form of an active male reproductive system and male secondary sex characters, along with a constantly arrested ovary. This intersexuality was exploited to follow changes caused by single gene silencing, accomplished via dsRNA injection. Cq-IAG silencing induced dramatic sex-related alterations, including male feature feminization, a reduction in sperm production, extensive testicular degeneration, expression of the vitellogenin gene, and accumulation of yolk proteins in the developing oocytes. Upon silencing of the gene, AG cells hypertrophied, possibly to compensate for low hormone levels, as reflected in the poor production of the insulin-like hormone . These results demonstrate both the functionality of Cq-IAG as an androgenic hormone-encoding gene and the dependence of male gonad viability on the Cq-IAG product. This study is the first to provide evidence that silencing an insulin-like gene in intersex C. quadricarinatus feminizes male-related phenotypes. These findings, moreover, contribute to the understanding of the regulation of sexual shifts, whether naturally occurring in sequential hermaphrodites or abnormally induced by endocrine disruptors found in the environment, and offer insight into an unusual gender-related link to the evolution of insulins.In sequential hermaphrodites, intersexuality occurs naturally, usually as a transition state during sexual re-differentiation processes. In crustaceans, male sexual differentiation is controlled by the male-specific androgenic gland (AG). An AG-specific insulin-like gene, previously identified in the red-claw crayfish  Drosophila melanogaster, gender-biased alternative splicing constitutes an important component of sexual differentiation Cherax quadricarinatus, the implantation of AGs into immature females led to the replacement of female characteristics with male traits, as well as to the cessation of vitellogenesis Understanding sexual shifts and differentiation in both vertebrates and invertebrates is of major interest, particularly given our changing environment. In vertebrates, such as fish, the regulation of such processes can be attributed not only to genetics but also to the effects of hormones and/or environmental factors C. quadricarinatus (Cq-IAG) and the freshwater prawn, Macrobrachium rosenbergii (Mr-IAG) The vast array of observed AG-associated effects has been attributed to an AG-borne hormone. Three orthologs of this putative AG hormone have been isolated from three species of isopods C. quadricarinatus, a gonochoristic species demonstrating distinct males and females, experiences non-transient intersexuality C. quadricarinatus undergo a dramatic morphological and physiological sex shift in which male reproductive organs regress, accompanied by ovarian activation and the onset of vitellogenesis AG hormone ineffectiveness, whether induced by AG ablation or resulting naturally from insufficient levels of secreted hormone(s), can account for hermaphroditism, as was suggested previously to explain part of a sexual shift in a protandric amphipod Although sexual shifts have been documented in invertebrates C. quadricarinatus, Cq-IAGCq-IAG in sexual differentiation has yet to be determined. Gene silencing using RNAi offers one strategy to address such questions. This approach has been successful in several crustaceans Cq-IAG in an intersex model to demonstrate an inducible sex shift. Specifically, male external sex characters were transformed to generate animals that exhibited female maternal care-related traits, along with testicular degeneration and AG hypertrophy (hAG). In parallel, ovarian activation was observed as the result of the onset of vitellogenesis. These findings confirm the pivotal role of Cq-IAG in the regulation of sexual differentiation in C. quadricarinatus. Significantly, elucidation of the role of this gene in C. quadricarinatus might contribute to understanding of the evolution of the control of processes regulating sexual shifts in protandrous crustaceans and may provide unique insight into the gender-related evolution of members of the insulin superfamily.The discovery of the first decapod AG-specific gene in Cq-IAG, accomplished via the construction of an AG cDNA library, has been described previously Cq-IAG ESTs from subsequent sequencing, showed that 115 out of 508 colonies were positive for Cq-IAG. This result, showing that Cq-IAG comprises approximately 25% of the ESTs, is with accordance with the previous screening of the library. Out of the non-Cq-IAG ESTs, 177 were randomly picked and sequenced. However, no new genes were identified and thus no further screening was conducted.The isolation of in-vivo assay demonstrating the functionality of Cq-IAG was to evaluate the effectiveness of gene knock-down via RNAi. For this purpose, 3 experimental groups were injected with either double stranded RNA (dsRNA) of the targeted gene, exogenous-based dsRNA or carrier only [doubly distilled water (DDW)]. The efficiency of injected Cq-IAG dsRNA in reducing Cq-IAG expression was revealed by real-time RT-PCR (Cq-IAG (0.24*105±0.16*105) were at least 30-fold lower   = 7.450, p = 0.0241 followed by multiple pair-wise comparison, p<0.05) than those of the control group, which was injected with DDW (8*105±2*105). The exogenous Mr-IAG dsRNA (4*105±1*105) showed a difference that turned out to be non-significant (p = 0.079) from that of the dsCq-IAG group, although a difference of at least one order of magnitude was observed in the raw data. This result showed that this silencing of Cq-IAG expression was specifically induced by Cq-IAG dsRNA injection, in a sequence-dependent manner.A prerequisite for a long e RT-PCR . SpecifiCq-IAG dsRNA injections had been established, a long-term silencing experiment addressing Cq-IAG functionality was performed, with two experimental young intersex groups- dsRNA-injected and DDW-injected. Silencing of the gene in young intersex animals induced the appearance of maternal care-related secondary sex characteristics. Comparing control intersex crayfish  gene is transcribed in the hepatopancreas, and its translated product is mobilized through the hemolymph to the ovaries, where it accumulates. Cq-IAG dsRNA injection induced both Cq-Vg transcription and expression of its encoded yolk protein (Cq-Vg expression was demonstrated by RT-PCR (C. quadricarinatus 18S rRNA (Cq-18S) housekeeping gene, which served as control for RNA extraction and the RT reaction. The 106-kDa vitellogenin polypeptide, indicative of secondary vitellogenesis, was detected and quantified in the hemolymph by using ELISA  could possibly also regulate AG activity, thereby inducing the observed hypertrophy, since hypertrophy of a gland in compensation for low levels of secreted product is well documented in cases of endocrine deficiencies An important observation of the present study is the extensive AG hypertrophy seen following C. quadricarinatus and its involvement in sexual regulation. Cq-IAG silencing in the intersex crayfish clearly revealed the involvement of this gene in maintaining maleness, as reflected in the empty vas deference and arrest of spermatogenesis that resulted from knock-down of Cq-IAG levels. Moreover, the extensive degeneration of the testicular lobules observed indicates that Cq-IAG is essential for male germ cell survival. Such involvement of an insulin-like peptide in testicular germ cell survival has also been documented in rats C. quadricarinatus individuals, male regression occurred simultaneously with ovarian activation and the onset of vitellogenesis, suggesting that a prerequisite for the activation of femaleness is a lack of sufficient levels of the AG factor, which in turn leads to apoptosis and degeneration of the male gonad and sexual shift. The results of this study, which are in accordance with previous ones A. vulgareThis study rests on the sexual plastic model of intersexuality as the basis for elucidating the role of the AG-specific insulin-like gene in The sexual shift induced in this study upon manipulation of a single AG gene may provide, for the first time, insight into the mechanisms underlying sexual shifts that occur naturally in many sequential hermaphrodites in the animal kingdom The building of a comprehensive picture of the processes of natural sexual shift opens the window for study of abnormal processes of sexuality. Such cases can be induced by man-made environmental pollutants that act as endocrine disruptors C. quadricarinatus AG cDNA SSH library Cq-IAG-repeated sequencing, four plates containing 127 colonies each (each cloned with a single EST from the described library) were screened, using a colony hybridization method Cq-IAG cDNA probe was synthesized using a random priming labeling mix . The probe was hydrolyzed to reduce its length to ∼200 b, as described in the DIG Application Manual (Roche Applied Science). The hydrolyzed probe mix was then hybridized to clones that had been fixed to nitrocellulose membranes. The membranes were hybridized over night, washed, sealed and exposed to BioMax MS Kodak film as described Cq-IAG were picked to liquid LB, grown over night (Qiagen DirectPrep 96 Miniprep) and sequenced as previously described The previously constructed C. quadricarinatus animals , mature males and females (40–70 g) of that species were also maintained in aquaria of the same size. The mature males had been subjected to surgical removal of the X-organ sinus-gland, an endocrine manipulation causing hypertrophy of the AG, as previously described ad libitum three times a week. Animals' weights were measured using analytical scales with a ±0.01 g error.Intersex Cq-IAG open reading frame (ORF) (accession no. DQ851163) was digested alternatively by NdeI and XbaI restriction enzymes , thereby yielding linear templates for sense and antisense Cq-IAG, respectively. Upon digestion, a small aliquot of each template was examined for digestion efficiency on a 1.3% agarose gel; linearized vectors were purified by the standard phenol:chloroform protocol and ethanol precipitation. Single-stranded RNAs were synthesized based on the above-mentioned linearized plasmids with a MEGAscript T7 kit  according to the manufacturer's instructions. Sense and antisense RNA were purified and hybridized as described previously in-vivo assay was synthesized based on the ORF of Mr-IAG, as previously described Mr-IAG encodes an orthologous peptide, its nucleotide sequence shows no significant similarity to Cq-IAG ; thus, it was chosen as a negative dsRNA control.The pGEM-T easy vector  including the Cq-IAG dsRNA-injected (n = 6), Mr-IAG dsRNA-injected (n = 6) and DDW-injected (n = 6). The dsRNA-injected animals were injected with 2 µg of dsRNA/g animal into the sinus of the fifth walking leg For a preliminary short-term silencing experiment, young male crayfish (15–17 g) were divided into three treatment groups, as follows: Cq-IAG-injected (n = 8) and DDW-injected (n = 7). The same procedure as the preliminary experiment was applied for the long-term assay, with the exception that the injections were given biweekly over a period of 25–30 weeks (2–5 molts from the beginning of the in-vivo assay).For the long-term silencing experiment, a group of 15 intersex individuals (each weighing 1–5 g) were selected and assigned into two groups: dsin-vivo experiment. Total RNA was isolated with the EZ-RNA Total RNA Isolation Kit, used according to the manufacturer's instructions . First-strand cDNA was synthesized by means of reverse transcriptase reaction using the Verso™ cDNA Kit (Thermo Fisher Scientific Inc.) with 1 µg of total RNA. RQ of Cq-IAG transcript levels were obtained using the following primers: IAG qPCR_F: 5′-GGCCTCCTCCCCTATCTGT-3′ and IAG qPCR_R: 5′-CCAGCCAGCAGCAGAATAGT-3′ with the FastStart Universal Probe Master (Rox) (Roche Diagnostics GmbH) and Universal ProbeLibrary Probe #144 (Roche). Cq-18S (accession no. AF235966), which was used as a normalizing gene, was also quantified by means of real-time RT-PCR using the primers: qCq-18S_F: 5′- CTGAGAAACGGCTACCACATC-3′ and qCq-18S_R: 5′-GCCGGGAGTGGGTAATTT-3′ with the above-mentioned mix and Universal ProbeLibrary Probe #74 (Roche). Reactions were performed with the ABI Prism 7000 Sequence Detection System .RNA was extracted from the AGs of males used in the short-term preliminary in vivo experimental animals, as mentioned above. The RT reaction was performed using M-MLV H minus reverse transcriptase (Promega), with 1 µg of total RNA from each sample according to the manufacturer's instructions. Cq-Vg (accession no. AF306784) was amplified by means of PCR with REDTaq ReadyMix™ PCR Reaction Mix  with the forward primer Cq-Vg F: 5′- AACGAGAGCCAGTCTTTGTGGCTG -3′ and the reverse primer Cq-Vg R: 5′- CAGCTTGTAGCTGTATGGACTACCAAG -3′ using cDNA obtained from hepatopancreas. Cq-18S served as the internal control for the RNA extraction and RT reaction. PCR products were separated on 1.3% agarose gel with Tris-acetate EDTA buffer, and bands were documented. AG cDNA was used to amplify Cq-IAG by PCR  with the reaction mix described above, using adaptor primers containing a gene-specific sequence flanked by a restriction recognition site and a T7 RNA polymerase sequence. The primers used were the forward T7-NdeI- F IAG: TAATACGACTCACTATAGGGTCTAGACTGATTGACTTCGACTGTGG -3′5′-  and reverse T7-XbaI- R IAG: TAATACGACTCACTATAGGGCATATGAACTGACGTAGATTCCGTCC-3′5′-. PCR products were separated as mentioned above. A fragment was excised, purified  and cloned into the pGEM-T easy vector (Promega). Clones containing the insert were isolated, and plasmid DNA was purified using HiYield Plasmid Mini Kit .RNA was extracted from samples of hepatopancreas and AG from mature males, from samples of hepatopancreas from mature females, and from the hepatopancreas and AG of the long-term The reproductive systems of the dsRNA-injected and DDW-injected intersex animals were dissected for morphological observation. Sperm ducts, testes, ovaries and AGs were fixed, dehydrated, embedded in paraffin and sectioned as previously described Escherichia coli cells, purified using a nickel column  and validated by MALDI-TOF. The confirmed recombinant Cq-IAG peptide was injected into a rabbit. The specificity of the antiserum was demonstrated by crossreactivity with the recombinant Cq-IAG by means of Western-blot. For the immunohistochemistry, paraffin sections of AGs obtained from endocrinologically induced For the immunodetection of Cq-IAG, a specific antibody was generated as follows: Briefly, a recombinant Cq-IAG peptide was expressed in The first left pleopod was removed from all intersex individuals at the beginning of the experiment, and the newly regenerated pleopod was removed at the end of the experiment. The widths of the endopod and the exopod were measured to calculate the EWI Data are expressed as means ± SEM. Due to the nature of the experiment the analyzed groups were relatively small and not normally distributed. Thus, non-parametric tests had to be used. All statistical analyses of a single parameter compared between 2 groups  were analyzed using the Mann Whitney U Test. The three and four groups analyzed in the real time RT-PCR and ELISA experimentation, respectively, were analyzed using the Kruskal Wallis Test followed by the correction of a multiple pair-wise comparison (built-in within the STATISTICA software) as accepted. We took into account that some groups might differ only in a marginally significant manner due to the usage of a multiple non-parametric test, which relies on a ranking principle.


Upon viral recognition, innate and adaptive antiviral immune responses are initiated by genital epithelial cells (ECs) to eradicate or contain viral infection. Such responses, however, are often accompanied by inflammation that contributes to acquisition and progression of sexually transmitted infections (STIs). Hence, interventions/factors enhancing antiviral protection while reducing inflammation may prove beneficial in controlling the spread of STIs. Serine antiprotease trappin-2 (Tr) and its cleaved form, elafin (E), are alarm antimicrobials secreted by multiple cells, including genital epithelia.We investigated whether and how each Tr and E (Tr/E) contribute to antiviral defenses against a synthetic mimic of viral dsRNA, polyinosine-polycytidylic acid (polyI∶C) and vesicular stomatitis virus. We show that delivery of a replication-deficient adenovector expressing Tr gene (Ad/Tr) to human endometrial epithelial cells, HEC-1A, resulted in secretion of functional Tr, whereas both Tr/E were detected in response to polyI∶C. Moreover, Tr/E were found to significantly reduce viral replication by either acting directly on virus or through enhancing polyI∶C-driven antiviral protection. The latter was associated with reduced levels of pro-inflammatory factors IL-8, IL-6, TNFα, lowered expression of RIG-I, MDA5 and attenuated NF-κB activation. Interestingly, enhanced polyI∶C-driven antiviral protection of HEC-Ad/Tr cells was partially mediated through IRF3 activation, but not associated with higher induction of IFNβ, suggesting multiple antiviral mechanisms of Tr/E and the involvement of alternative factors or pathways.This is the first evidence of both Tr/E altering viral binding/entry, innate recognition and mounting of antiviral and inflammatory responses in genital ECs that could have significant implications for homeostasis of the female genital tract. Genital epithelial cells (ECs) provide the first line of defense against sexually-transmitted infections (STIs) The physiological role of serine antiproteases has been extensively studied over the past two decades Viral double-stranded RNA (dsRNA) is a PAMP generated during the life cycle of most, if not all, viruses in vitro and in vivo, making polyI∶C an attractive candidate for microbicide or vaccine adjuvant trials against STIs et al. reported an anti-HIV feature of E in in vitro study et al. documented that Tr expression increased Ad/LacZ viral clearance, as well as secondary immune responses, in a murine model in vivoTreatment with polyI∶C has been shown to induce potent antiviral protection Collectively, these data clearly demonstrate the importance of Tr/E in antiviral protection, although specifically how Tr/E contribute to antiviral immune-inflammatory responses is still unknown. Thus, the objective of this study was to elucidate whether and how Tr/E contribute to innate antiviral and inflammatory responses in uterine ECs elicited by a viral ligand, polyI∶C. Here, we describe novel antiviral and immunomodulatory properties of Tr/E. To the best of our knowledge, this is the first study to present evidence of Tr/E affecting host innate recognition and modulating antiviral and inflammatory responses in genital ECs against polyI∶C.in vitro experiments and as reference markers for Western blotting. The amount of Tr/E being used in this study ranges from 0.2 to 5 µg/ml to cover the physiological levels (within 1 µg/ml) reported previously PolyI∶C and lipopolysaccharide (LPS) were reconstituted in the phosphate-buffered saline (PBS) and used at concentrations shown in figures . Human recombinant (r) proteins Tr (rTr)  2. Caco-2 cells were cultured in DMEM growth medium containing 5% FBS, 1% l-glutamine, 1% penicillin-streptomycin, 5 mL NEAA, and 9.6 mL NaHCO3.HEC-1A, Caco-2 , and A549  cells in vitro studies. For routine experiments, epithelial cells were either treated with Opti-MEM medium alone (UT) or with MOI 50 PFU of Ad/dl or Ad/Tr at 37°C overnight. After PBS washes and rest for 4 h, cells were incubated in serum-containing medium alone or with polyI∶C for additional 24 h.The Ad constructs used in this study have been described in detail elsewhere Vesicular stomatitis virus (VSV-GFP), a lytic IFN-sensitive virus expressing green fluorescent protein under the viral promoter , was used in a plaque reduction assay MTT assay, described elsewhere VeriKine™ Human Interferon-Alpha Multi-Subtype ELISA Kit  according to the supplier's protocol. Analytes were quantified based on standard curves obtained using an ELISA reader Tecan Safire ELISA reader (MTX Labs Systems Inc.). Cut off limit for Tr/E and IL-8 was 31.25 pg/ml; for TNFα and IFNβ was 15.6 pg/ml; for IFNα subtypes was 12.5 pg/ml, and levels detected below these limits were considered as undetectable.Cell-free supernatants were stored at −70°C until assayed for human Tr/E, IL-8, TNFα, IL-6 with ELISA Duoset kit (R&D Systems), human IFNβ by ELISA kit from Antigenic America Inc. , and IFNα subtypes by 5′-ACAGGATCCGCGCAAGAGCCAGTCAAAGGTCCA-3′ and: 5′-CAGGAATTCTCACTGGGGAACGAAACAGGCCATC-3′. The amplified elafin-cDNA was gel purified, restriction digested, and directionally subcloned into BamH I and EcoR I site of bacteria expression vector pHAT10  in frame to the HAT-tag. The clone was confirmed by restriction digestion and nucleic acid sequencing of both strands. The plasmid was transformed into Escherichia coli BL21 strain . When the cells grew in Luria-Bertani broth containing 100 µg/ml ampicillin to an A600 of 0.55–0.60 at 37°C, protein expression was induced by adding isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 1 mM for 6 h. The cells were harvested by centrifugation at 2,000×g for 5 min, and cell pellets were washed once with ice-cold PBS (pH 7.4) and resuspended in wash buffer . The desired recombinant protein, rE, was found in the soluble fraction in pilot experiment. After disrupting the cells by freeze-thaw and subsequently by sonication, the lysates were centrifuged at 20,000×g for 20 min, and the soluble fraction was collected. The soluble recombinant protein was purified from this fraction by BD TALON metal affinity resin  according to the manufacturer's instructions and dialyzed against PBS at 4°C. The expressed human rE was confirmed by Western blotting targeting the HAT-tag as well as by specific antibody for human E.To prepare rE protein, cDNA fragments encoding the relevant part of Tr (amino acid residues A61–Q117) were amplified from HEC-1A cells cDNA using the following primers: Whole-cell extracts were prepared by using whole-cell extract buffer ) as per standard protocol. Protein amount was quantified using Bradford assay with bovine serum albumin (BSA) (Sigma-Aldrich) as a standard and Bio-Rad Dye Reagent Concentrate as a protein stain . WB was performed on a 10% polyacrylamide denaturing SDS-PAGE gel and PVDF membranes  as per standard protocol, using the following primary antibodies: anti-human Tr/E TRAB2O , tIRF3 FL-425 , pIRF3 Ser396 , RIG-I #4520 , MDA-5 R470 , c-Jun (60A8) , p-c-Jun (Ser73) , TLR3 IMG-5631-2 , NF-κB p65 (C-20): sc-372 (Santa Cruz), GAPDH ab9485 (Abcam) antibodies at a dilution of 1∶1000, except for GAPDH (1∶5000) and p-NF-κB p65 (Ser 536): sc-33020 (Santa Cruz) (1∶100). After incubation with corresponding horse-radish peroxidase (HRP)-conjugated secondary antibody (Bio-Rad Laboratories), blots were visualized using a SuperSignal West Femto or Pico Chemiluminescent Substrate kit . GAPDH was used as internal loading control. Quantification of band intensities was done using MBF_ImageJ for Microscopy Software.5 per well) were transfected in 0.5 mL of Opti-MEM® medium (Invitrogen Life Technologies) in a 24-well plate with 20 ng of DNA of each of pgkβ-Gal and pNF-κB-Luc or pAP-1-Luc  plasmids  and 50 MOI of Ad/dl or Ad/Tr overnight at 37°C utilizing Lipofectamine 2000 (Invitrogen Life Technologies) as per supplier's instructions. Pgk-β-Galactosidase plasmid was used as a normalization control. After transfection, cells were extensively washed with PBS and allowed to rest for 4 h followed by stimulation with polyI∶C and LPS for 2–4 h. After ligand stimulation, cells were washed twice with cold PBS, lysed with 1× reporter buffer  and subjected to one freeze-thaw cycle, after which cell lysates were collected and assayed in a 96-well plate for luciferase  and β-galactosidase  activities separately as per supplier's protocol, using Opticomp I luminometer (MGM instruments).HEC-1A cells , 12935-300, (Invitrogen Life Technologies) were used to specifically knockdown IRF3. Transfections of siRNA (8 pmol) were done using Lipofectamine RNAiMAX and Opti-MEM® I Reduced Serum Medium (Invitrogen Life Technologies) as per supplier's instructions. HEC-1A cells, 3×104 in a 100 µl of total volume of complete growth medium, were transfected in a 96-well BD Falcon culture plate (BD Biosciences) for 48 h before adding MOI 50 PFU of Ad/dl or Ad/Tr. Knockdown efficiency was monitored using WB.Small interfering (siRNA) molecule (Invitrogen Life Technologies) targeting IRF3 (GenBank accession number NM_001571) within 247–1530 ORF through the following sequences: IRF3-498 (start) 5′-AGGGCTTTACAAATCCTGCTCTCTTCA-3′ (probe), 5′-GGTGTTCCAGATGCCAGAC-3′ (forward), 5′-TTCCGCAAATGTGAAGTGTATAA-3′(reverse); MDA5: 5′-TTTGGCTTGCTTCGTGGCCC-3′(probe), 5′-TGATTCCCCTTCCTCAGATAC-3′(forward), 5′-TGCATCAAGATTGGCACATAGT-3′(reverse); TLR3: 5′-TGTGGATAGCTCTCC-3′(probe), 5′-CCGAAGGGTGGCCCTTA-3′(forward), 5′-AAGTTACGAAGAGGCTGGAATGG-3′ (reverse); 18S rRNA: 5′-CGGAATTAACCAGACAAATCGCTCCA -3′ (probe), 5′-GTGCATGGCCGTTCTTAGTT-3′ (forward), 5′-TGCCAGAGTCTCGTTCGTTAT-3′ (reverse). The expression of 18S ribosomal RNA (rRNA) was used as an internal control. PCR was run with the standard program: 95°C 10 min, 40 times of cycling 95°C 15 sec and 60°C 1 min in a 96-well plate with an ABI PRIZM 7900HT Sequence Detection System using the Sequence Detector Software 2.2 (Applied Biosystems). To determine the expression of ISG56, a semi-quantitative RT-PCR was performed with oligonucleotide primer sequences (Mobix): 5″-GACAGGAAGCTGAAGGAGAAA-3′ (445-bp product) (forward), 5′-TcTTGCATTGTTTCTTCTACCACT-3′ (reverse). PCR program was as follows: 94°C for 2.5 min, 30 cycles of 94°C for 20 sec, 55°C for 30 sec, 72°C fir 1 min, 72°C for 5 min. PCR products were electrophoresed on 2% agarose gel using a 100-bp DNA ladder (Invitrogen Life Technologies) as a marker to identify PCR products. The gel was stained with a loading fluorescent dye EZ-vision N472-Q  and visualized with UV transilluminator . Densities of DNA bands were quantified to signal volumes using ImageQuant 5.0.The protocol for RNA isolation was described elsewhere Immunofluorescence staining was performed as described earlier N-methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide (Sigma-Aldrich), diluted to 50 µg/ml in 50 mM Tris, 0.1% Triton, 0.5 M sodium chloride, pH 8 buffer were added and the hydrolysis was recorded by monitoring the increase of absorbance at 405 for 15 min using a Tecan Safire ELISA reader (MTX Labs Systems).This assay was performed by measuring inhibition of HNE activity by Tr/E as was described earlier t test or a one-way analysis of variance (ANOVA) using Sigma Stat 2.03.Data were expressed as means ± standard deviation (SD). Statistical analysis was performed with either unpaired Student's Initial studies evaluated the expression of the secreted Tr from Ad/Tr-cells by ELISA that detects both Tr/E Being foremost a protease inhibitor, Tr in Ad/Tr-sups was tested in protease inhibition assay Since polyI∶C stimulation of ECs triggers the induction of antiviral protection We next attempted to elucidate specific mode(s) of Tr/E antiviral activity. Given the possibility of additional Tr/E being released from Ad/Tr-cells after polyI∶C stimulation and upon VSV-GFP challenge and thus potentially acting on both virus and cells, we tested if both or each Tr/E individually were acting through two separate mechanisms: (i) direct antiviral activity exerted during the virus/cell encounter and viral binding/entry, targeting either virus or cells; and (ii) indirect cell-associated immunomodulatory activity that targets polyI∶C-triggered cellular antiviral responses and protection. To minimize the Ad-associated effects, we used secreted/soluble proteins delivered to Ad-uninfected HEC-1A cells. Because we could not use Ad/Tr-sups after polyI∶C stimulation as a source of both Tr/E, since they would contain residual polyI∶C and thus mask the effect of Tr/E alone, a commercial rTr (with a C-terminus His-tag) was used as a source of both Tr/E, based on results shown in Next we assessed antiviral properties of individual secreted/soluble Tr/E preparations. We show that VSV-GFP replication was reduced  in Ad/Tr-sups-treated cells , compareConsidering that polyI∶C can induce apoptosis We next elucidated the immunomodulatory effect of Tr/E in Ad/Tr-cells in context of polyI∶C stimulation. Type I IFNs are considered a hallmark of antiviral response, with IFNβ being a key correlate of polyI∶C-induced antiviral protection in vitroPhosphorylation and nuclear translocation of IRF3 are key events in the transcriptional activation of inducible ISG cellular genes ISG15 and ISG56 have been implicated in antiviral protection and linked to activation of IRF3 in response to polyI∶C We next elucidated phosphorylation and activation of other transcription factors, including NF-κB and c-Jun, that have also been shown to contribute to antiviral responses PolyI∶C-induced antiviral protection of primary genital ECs was previously associated with heightened expression of TLR3 We next determined whether IRF3 was required for enhanced polyI∶C-driven antiviral state in Ad/Tr-cells. IRF3 was knocked down by utilizing IRF3-specific small interfering RNA and cells were subsequently treated, or not, with polyI∶C before challenging with VSV-GFP. The greatest knockdown efficiency was observed between 72 h and 96 h post transfection (data not shown). Results of WB demonstrated no apparent expression of tIRF3 after siRNA treatment . FurtherPolyI∶C stimulation induces not only antiviral, but also pro-inflammatory factors WAP proteins, including antiproteases Tr/E, SLPI, and ps20, are pleiotropic molecules known to play multiple and significant roles in health and disease This is the first study that comparatively assessed the presence and potential mechanisms of antiviral activity of each Tr/E. Here, we presented evidence showing two distinct, but likely complimentary antiviral properties of Tr/E: (i) direct antiviral activity exerted during the virus/cell interaction and targeting virus, but not cells; and (ii) indirect and cell-associated immunomodulatory activity, targeting polyI∶C-triggered cellular antiviral responses. The virus-mediated activity was observed in the absence of polyI∶C stimulation and in the presence of Tr in Ad/Tr-cells (presumably present in both Ad/Tr-sups and Ad/Tr-cells). Because only Tr was detected in polyI∶C-untreated Ad/Tr-cells, and because the expression of IFNβ was low and not different between untreated Ad/dl and Ad/Tr groups, we conclude that in the absence of polyI∶C, Tr alone was mediating antiviral activity in Ad/Tr-cells by acting either directly on virus or indirectly through cells. However, we failed to transfer this direct antiviral effect of Tr via Ad/Tr-sups, possibly due to the absence of an additive protective effect from augmented intracellular Tr (as would be expected in Ad/Tr-cells),or due to an inefficient delivery of Tr in supernatants and a “diluting" effect from other antiviral factors released in response to Ad/dl delivery , insert.et al. findings, describing a direct antiviral effect of E as a mode of action against HIV Additionally, the presence of both Tr/E, as in rTr preparation, was also protective against VSV-GFP challenge in the absence of polyI∶C treatment. Although no reports describing direct antiviral activity of Tr are published to date and no precise mechanisms of Tr/E direct antiviral effect have been identified, our observation with rTr is in line with Ghosh et al. also reported that primary uterine EC produced Tr/E in response to polyI∶C Our results further suggest that the presence of both Tr/E might be required for each virus- (direct) and cell-associated (indirect) antiviral effect, as was evident from our data using Ad/Tr-cells, Ad/Tr-sups, and rTr, indicating that perhaps the most efficient antiviral protection depends on collaborative work of both Tr/E. Interestingly, when HIV-susceptible, but uninfected, CSWs were followed prospectively, those who remained HIV-negative had elevated levels of both Tr/E detected in CVLs et al. supports this argument, since they showed that the N-terminus of Tr had a better affinity for LPS than its C-terminus end It is unclear why tested rE failed to show antiviral activity against VSV-GFP; it could be attributed, however, to a HAT-tag insertion at the N-terminus of rE. Indeed, all rTr, rE, and secreted Tr were equally functional against HNE (data not shown) and capable of inhibiting IL-8 production in response to polyI∶C . Yet, whThe second antiviral property of Tr/E observed in our study was an indirect cell-mediated immunomodulatory activity of Tr/E, targeting polyI∶C-induced antiviral cellular responses. In contrast to responses to bacterial or pro-inflammatory stimuli in vitro in response to LPS and polyI∶C, respectively. These observations further support our results and strengthen the earlier argument of Tr/E acting either directly against VSV-GFP or through cells and additional factors/pathways in Ad/Tr-cells. Furthermore, moderation of immune-inflammatory responses and thus curbing undesirable immune activation might be one of the protective mechanisms of innate antimicrobials at mucosal sites. Additionally, in searching for ISGs typically associated with antiviral protection and IRF3 activation Our results showed that polyI∶C-triggered antiviral cellular protection was significantly enhanced in the presence of Tr (in Ad/Tr-sups) and Tr/E (in Ad/Tr-cells and in rTr). We also showed that polyI∶C-mediated activation of IRF3 was further induced in Ad/Tr-cells, compared to controls, whereas IFNβ expression was dampened. Interestingly, human β defensin 3 et al. showing that a knockdown of ISG56 was associated with increased IRF3 activation and inhibition of VSV-GFP replication This is the first report on the involvement of serine antiproteases, Tr/E in particular, in antiviral signaling pathways. As no prior data are available on the role of Tr/E in IFNβ and IRF3 induction, further and more detailed investigations might be required to explain why in Ad/Tr-cells IFNβ and ISG56 levels were reduced while IRF3 activation was increased. We hypothesize, however, that this phenomenon could be an attempt of Tr/E to control antiviral inflammatory events through RIG-I/MDA5 and NF-κB downregulation while increasing cellular protection through activation of IRF3 and/or alternative factors or pathways. Although most of the studies show ISG56 to be associated with upregulated IRF3 Inflammation is one of the leading factors predisposing to acquisition and disease progression of STIs in the FGT The precise mechanism(s) of immunomodulatory effects of Tr/E, as both secreted and/or intracellularly expressed proteins, on viral sensors, cytokines, and IFNβ in response to polyI∶C treatment is still largely unknown. This is partly because the existence of the cognate receptor for Tr/E remains elusive, and it is equally unknown whether Tr/E require a receptor to function. We propose that reduced levels of IL-8, IL-6, TNFα and IFNβ in Ad/Tr cells in response to polyI∶C are likely a result of overall attenuation of RIG-I and MDA5 levels, as they are known to regulate the expression of pro-inflammatory and antiviral mediators et al. showed that SLPI Gly(72) mutant, unlike other mutants tested in that study, lost its in vivo immunosuppressive activity against NF-κB activation and neutrophil recruitment in the lungs that appeared to be most closely related to SLPI's trypsin-inhibiting activity A noteworthy observation of this study is that while Tr from Ad/Tr-sups and rE were found functional against HNE and able to inhibit polyI∶C-induced IL-8 production, they did not show any antiviral activity, suggesting that antiprotease, anti-inflammatory, and antiviral activities of the tested proteins may not necessarily be co-dependent or predictive of each other; nonetheless, they can be complimentary. This observation is supported by earlier reports, showing both a protease non-inhibitory N-terminus and an inhibitory C-terminus of Tr exhibiting comparable antibacterial and antifungal functions Overall, our data support and further extend earlier observations on immunomodulatory effects of Tr/E


To investigate the outdoor aeroallergens at two different, a fully developed (FD) and a less developed (LD), sites in Riyadh, capital city of Saudi Arabia.Two Burkard Volumetric 7-day Recording Spore Traps were employed at both locations. Exposed slides were read at 12 bi-hourly traverses, with a total of 60 fields per 24 hours (0-24hours). The study was initiated in January 2012.Phoenix dactylifera (31%), grass pollen (14%), Atriplex nummularia (12%), Amaranthus viridus (10%), Artemisia monosperma (7%), Plantago sp. (6%) and Populus sp. (4%). The minor species (<1%) were Casuarina sp., Chenopodium murale, Eschscholtzia sp., Eucalyptus sp., Lilium sp., Prosopis juliflora, Salix sp. and Salsola imbricata. At the LD site, the major species included Phoenix dactylifera (36%), grass pollen (15%), Salix sp (8%), Artemisia monosperma (6%), Atriplex nummularia (6%), Plantago sp. (3%), Populus sp. (3%), Amaranthus viridus (2%) and Ambrosia sp. (2%). The minor species included Daucus carota, Eucalyptus sp. and Salsola imbricata. The fungal spores recorded at both sites displayed only quantitative variations. The major components of these spores at FD site included Arthrinium (14%), Ulocladium (14%), Cladosporium (13%), Alternaria (10%), ascospore septate (10%), smut spores (9%), Basidiospore (7%), ascospore non septate (6%), Bipolaris (3%), Myxomycete (3%), Periconia (3%), Aspergillus/Penicillium (2%), Stemphyllium (2%) and Curvularia (1%). The minor components (<1%) were Drechslera, Melanospora, Peranospora, Pithomyces and Torula. The major fungal spores at LD site were Ulocladium (13%), Cladosporium (12%), Aspergillus/Penicillium (11%), smut spores (11%), Alternaria (9%), ascospore septate (9%), Basidiospore (9%), Arthrinium (8%), Stemphyllium (5%), ascospore non septate (4%), Periconia (3%) and Bipolaris (2%), while the minor spores included Curvularia, Drechslera, Melanospora, Myxomycete, Peranospora, Pithomyces and Torula.The data analyzed so far revealed the presence of different pollen and fungal species at both sites. The ratio between the pollen and fungal spores was 1:5. The airborne pollen grains showed quantitative and qualitative differences between the two sites. At FD site, the major pollen grains included The results indicate that developed and less developed sites have no impact on the qualitative and quantitative diversity of fungal spores but such diversities were recorded in airborne pollen species.


Hearing impairment substantially affects child's ability to normally acquire the spoken language. Such negative effects create problems for the child not only in terms of communication but also in terms of achievement in school as well as social and emotional growth. The aim of this research is to study the prevalence of hearing disorders and its relationship to age and gender among primary school students of Zahedan, Iran. In this cross-sectional and descriptive analytical study, 1500 students from elementary schools were screened for hearing loss. The selection of samples was performed using multistage sampling method. Primary information was obtained through direct observation, otoscopy, and audiometric and tympanometric screenings. Data was obtained and analyzed via ANOVA test. Statistical analysis showed a significant correlation between the age and the prevalence of middle ear abnormal function. Conductive hearing loss in males and females was 8.8% and 7.1%, respectively. In addition, 1% and 0.7% of male and female students, respectively, suffered from sensorineural hearing loss. Results indicated that 20.2% of students of elementary schools in Zahedan needed medical treatment for their problems. Therefore, it is recommended that the hearing screening of school-age children should be included in annual school health programs in this region. The program should be run annually for children aged 3–9 years. After nine years of age, the program should be performed annually for children at risk. Hearing loss of even 15 dBHL can create hearing disability in children and consequently impairment in their mental growth . Due to Presence of any structural and anatomic problems of auricle and the external ear canal. Detection of abnormal cases of the ear canal and eardrum at the time of otoscopic examination. No response of either ear to at least one of the experimental frequencies. Detection of type B or C tympanogram. In this study, the MT10 Audiometry and Tympanometry screening device was used for primary screening. The research results were collected by special forms, and the frequency distribution of different hearing impairments was obtained. Chi-square test was also used to compare the differences between both genders, and relationship between hearing impairment and students' age was evaluated by ANOVA test.In this cross-sectional and descriptive analytical study, 1,500 students from 30 elementary schools of Zahedan in the academic year 2010-2011 were screened for hearing loss. The selection of samples was performed through multistage sampling method. Out of 146 elementary schools in Zahedan, 30 schools were selected as follows: the schools of the city of Zahedan were divided into six regions based on two educational districts and 3 municipalities, and some schools were randomly selected from each region based on the relative frequency of the schools of that region. From each school, 50 students in 5-year range (6 to 10 years old) were selected randomly (10 persons from each age). In total, 300 males and females per age were included in the study. Physical environment in schools such as noise levels (inside and outside classes) was within the permissible level, and according to ANSI S12.602002, and weather temperature during testing was between 28 and 33°C. Primary information was obtained through direct observation, otoscopic examination, and audiometric and tympanometric screenings of every student. The rejected subjects were referred to for the full examination of hearing system that included complete medical history, pure tone and speech audiometric tests, and tympanometric as well as acoustic reflex assessments. The inclusion criteria for the specialized evaluation included the following. z = 3.9057). Other notable result of the tympanometry screening was the significant decrease of negative pressure incidences in the ear with an increase in age , which suggests the decrease of type C tympanogram incidences with an increase in age. A particular correlation coefficient was obtained for the relationship between the amount of conductive problems cases and school grade of students at estimated value of r = −0.972, degree of freedom of df = 3, and confidence coefficient of t = 19.331, respectively. In other words, conductive problems are reduced with grade increase  [% [9] andThe results of this study had some similarity with behavioral test results of Poland study . In IranHearing loss and its consequent difficulties on speech and language might be controlled and treated via appropriate hearing screening protocol and program in every educational setting; it is noteworthy that, according to the obtained results, the authors emphasize annual hearing screening programs for school-age children in order to promote health care and to prevent social and educational problems in this region, as these programs are being carried out in other areas.


Hearing impairment is common in patients with mitochondrial disorders, affecting over half of all cases at some time in the course of the disease. In some patients, deafness is only part of a multisystem disorder. By contrast, there are also a number of “pure” mitochondrial deafness disorders, the most common probably being maternally inherited. We retrospectively analyzed the last 60 genetically confirmed mitochondrial disorders diagnosed in our Department: 28 had bilateral sensorineural hearing loss, whereas 32 didn't present ear's abnormalities, without difference about sex and age of onset between each single group of diseases. We reported also a case of MELAS patient with sensorineural hearing loss, in which cochlear implantation greatly contributed to the patient's quality of life. Our study suggests that sensorineural hearing loss is an important feature in mitochondrial disorders and indicated that cochlear implantation can be recommended for patients with MELAS syndrome and others mitochondrial disorders. Mitochondrial diseases are disorders caused by impairment of the mitochondrial respiratory chain. The genetic error can affect both mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) .MtDNA mutations are classified as either large-scale rearrangements , usually sporadic, or point mutations, which are usually maternally inherited, and concern genes responsible for protein synthesis (rRNAs or tRNAs), or genes encoding subunits of the electron transport chain (ETC) . The pheHearing impairment is common in patients with mitochondrial disorders, affecting over half of all cases at some time in the course of the disease . AlthougBy contrast, there are also a number of mitochondrial “pure” deafness disorders, the most common probably being maternally inherited deafness due to the A1555G mutation in the 12 s rRNA gene, MTRNR1 . The heaThe pathological examination of the inner ear is technically demanding, highly specialized, and only possible postmortem. There have, therefore, only been a few detailed pathological studies of the auditory system in patients with mitochondrial diseases. In the cochlea, the stria vascularis maintains the ionic gradient necessary for sound transduction and the complex interaction between inner and outer hair cells . These cHearing loss is usually peripheral (due to cochlear or auditory nerve dysfunction), but in patients with a multisystem mitochondrial disorder, the auditory system may be affected at the brain stem, midbrain or at a higher level in the auditory cortex. The peripheral hearing loss typically affects high frequencies first, followed by intermediate frequencies, and finally involving low frequencies and causing the typical “flat” audiogram seen in a severely deaf individual. The preferential involvement of high frequencies may be related to the relatively high energy requirements of the basal cochlea . The vasIn this review, we analyzed the results of a retrospective study about the presence of hearing loss in a cohort of patients with genetically confirmed mitochondrial disease and we described the results and follow-up of a MELAS patient who underwent cochlear implantation.We retrospectively analyzed the last 60 genetically confirmed mitochondrial disorders diagnosed in our Department in order to identify patients affected with hearing loss. Males and females were quite equally distributed , with an age at diagnosis ranging from 8 to 73 years; the mean age of this group of patients was 45 years old. Three patients belonged to the same family.Most of patients were referred to our centre for a suspected mitochondrial disease and underwent a muscle biopsy (quadriceps or deltoid muscle). In these cases, DNA was extracted from frozen muscle tissue and used for direct sequencing of mitochondrial and/or nuclear genes, with standard methods , 20. In mtDNA point mutations were searched by screening the whole mtDNA using the MitoScreen Assay Kit for the Transgenomic WAVE System and DHPLC (denaturing high-performance liquid chromatography) with single-stranded conformational polymorphism analysis, following supplier's indications or by using the ABI PRISM BigDye Terminators v3.0 Cycle Sequencing Kit.Presence of single or multiple deletions of mitochondrial DNA was revealed by long-range PCR or southern blot analysis, using standard methods . mtDNA pAll of the cases included in this study performed audiometric examination before to receive the genetic diagnosis. The cut-off of hearing loss was defined according to the mean hearing loss at frequencies of 250, 500, 1000, 2000, and 4000 Hz as follows: normal hearing ≤ 20 dB hearing loss.Out of the 60 cases, 28 had bilateral sensorineural hearing loss, whereas 32 did not present ear's abnormalities. Clinical findings and the frequency of SNHL are summarized in One of the 28 patients with hearing loss, a MELAS patient, was submitted to cochlear implantation. Following, we briefly reported his main clinical notes.He was a 46-year-old man who started to complain mild bilateral hearing loss from his 20; he had normal prenatal and perinatal histories. His family history was also normal. Diabetes mellitus was noted at the age of 33, and insulin therapy was initiated. Serum biochemical studies showed a high level of lactic acidosis, which increased after exercise, and echocardiography disclosed the presence of hypertrophic cardiomyopathy. Neurological examination showed diffuse mild muscle weakness, more evident in distal lower limbs, with stepping gait. Computer tomography (CT) and magnetic resonance imaging (MRI) scans showed no abnormality in either inner ear. MRI demonstrated mild cerebellar atrophy.Muscle biopsy revealed strong reactive vessels on Succinate Dehydrogenase stain (SDH), without ragged red neither citochrome C-oxidase negative fibers. Molecular analysis revealed the presence of the A3243G mitochondrial DNA heteroplasmic mutation.During next years, hearing loss markedly worsened: pure-tone audiometry revealed bilateral profound hearing loss with an average of 130 dB in both ears. The hearing aid was no longer useful. In a promontory stimulation test, the patient responded to electrical stimulation in both ears at the intensity of 0.3 mA.At 33 years old, he underwent cochlear implantation surgery using a 24-channel device (cochlear-contour) in the right ear. Twenty-five electrode rings were successfully inserted and all 22 channels and 2 extracochlear electrodes were found to be usable at the initial “switch on” of the cochlear implant.Clinical followup was quite stable: he referred sometimes episodes of gastrointestinal pseudoobstruction and few periods of scarce glycemic control; outcome of cochlear implantation was good: postoperative neural response telemetry (NRT), implant-evoked BAEP and middle latency response (MLR) showed good responses. Eight years after the surgery, the patient could use the telephone and was satisfied with the improvement in communication due to the cochlear implant.Hearing impairment is a common feature of mitochondrial disease, either in isolation, or as a part of a complex multisystem disorder, with the cochlea bearing the brunt of the pathology. Ultimately, all forms of mitochondrial deafness arise through a respiratory chain defect causing ATP depletion, but it is not clear why hearing should be preferentially affected in some mitochondrial disorders and not in others, nor why cochlear pathology can vary between different disorders. There is clear evidence of a major environmental influence in some forms of mitochondrial deafness, and the interaction between nuclear and mitochondrial genes appears to be important .The degree of hearing loss correlates well with the mutation load in skeletal muscle , and theThe percentage of mutated mtDNA undoubtedly contributes to the clinical variability seen among patients, but this does not provide the whole explanation. It is not currently known why certain maternal pedigrees transmitting A3243G tend to develop a pure deafness-diabetes phenotype, whereas others only show ptosis and external ophthalmoplegia, and yet others are affected by severe multisystem MELAS phenotype . AdditioOur study considers only the syndromic form of mitochondrial SNHL, showing that the frequency of hearing loss in our group of patients is the same of the most of studies reported in literature , 21; morHowever, the study offers important considerations, like the relative low frequency of hearing loss in patients with chronic progressive external ophthalmoplegia (CPEO) (16%), which could be considered in most of cases a “pure” myopathy, and, by contrast, the high frequency of hearing loss in patients affected by mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) (75%), in which the diagnosis may be difficult at the beginning and the presence of hearing loss could orient clinicians in considering a mitochondrial disease in the differential diagnosis .The case presented in this study remarks the importance to consider cochlear implant in patients with mitochondrial SNHL. The patient suffered from hearing loss due to MELAS syndrome; after cochlear implantation his quality of life markedly improved and he could preserve his work.Since the first recorded cochlear implant in a patient with Kearns-Sayre syndrome , many paIn conclusion, individuals who harbour mtDNA mutations may be at risk of developing severe hearing deficit, and these individuals should avoid ototoxic agents, such as aminoglycoside antibiotics, which may further compromise cochlear function .Our study suggests that SNHL is an important feature in mitochondrial disorders and should be considered in the diagnostic workup and management of patients with suspected mitochondrial disease. We found that cochlear implantation in a patient with multisystem degenerative disease greatly contributed to the patient's quality of life and made it possible for him to communicate with family members and caregivers better than previously.The results indicated that cochlear implantation can be recommended for patients with MELAS syndrome and other mitochondrial disorders, if they have residual retrocochlear function.


Current evidence supports the central role of neuropeptides in the molecular mechanisms underlying dental pain. In particular, substance P, a neuropeptide produced in neuron cell bodies localised in dorsal root and trigeminal ganglia, contributes to the transmission and maintenance of noxious stimuli and inflammatory processes. The major role of substance P in the onset of dental pain and inflammation is increasingly being recognised. Well-grounded experimental and clinical observations have documented an increase in substance P concentration in patients affected by caries, pulpitis, or granulomas and in those undergoing standard orthodontic or orthodontic/dental care procedures. This paper focuses on the role of substance P in the induction and maintenance of inflammation and dental pain, in order to define future lines of research for the evaluation of therapeutic strategies aimed at modulating the complex effects of this mediator in oral tissues. A f. A f7]. A pronounced density of NK2 receptor has been detected in gingival and Malassez epithelium. NK1 and NK2 receptors were also shown on fibroblasts in the periodontal ligament and in the pulp . SubstanSensory fibers terminate near blood vessels, and SP receptors are present in high concentrations in this tissue , 28. In SP interacts with mast cells and induces the release of histamine, therefore causing elevated vascular permeability and increased blood pressure . MoreoveAlmost all pathological conditions that affect oral tissues, as well as orthodontic or dental care procedures, increase the production and release of SP .ex vivo study have shown that SP expression is significantly greater in grossly carious painful molars than in grossly carious asymptomatic molars [The results of an c molars . Mean exc molars , 32. Intc molars .Considering that SP exerts its biological activity mainly via the high affinity NK1 receptors but that at higher concentrations the peptide can activate also NK2 and NK3 receptors, it can be speculated that while in physiological condition NK1 is the most involved receptors, in pathological conditions, due to the higher SP concentrations, also NK1 and NK2 become involved.It has been suggested that SP innervation and SP-related neurogenic inflammation could play a role in several orthodontic procedures.Deep cavity preparation (<1 mm remaining dentine thickness) for orthodontic reasons was associated with a significant increase in the concentration of SP in pulp tissue, compared with controls . It is wA recent study has also shown that dentin bonding agents used in restorative dentistry can moderately increase SP release in the dental pulp. This effect is likely to be a result of reactive oxygen species that are formed, which have the ability to diffuse through dentinal tubules, constituting a biological risk to the pulp. Because cavity preparation and the use of dentine bonding agents cooccur very often, a final significant alteration of SP concentrations is likely to occur, that participates in neurogenic inflammation .Similarly, an increase in SP expression and release was observed in patients undergoing orthodontic tooth movement, which was associated with enhanced synthesis of proinflammatory cytokines , 37.Interestingly the kinetic of SP release parallels the perception of pain during orthodontic treatment, which suggests that the perception of pain may be linked to the release of SP –40.Further studies have analyzed the impact of different bracket systems on SP concentrations, with the aim of reducing the inflammation and pain resulting from these procedures .A high level of SP expression has also been reported following procedures with a low impact on tooth physiology, such as light- and laser-activated tooth bleaching .Although it is not possible to reach a conclusion about the biological role of SP release during orthodontic movement, it can be suggested that its release as a consequence of the stimulation of peripheral nerve terminals by means of orthodontic forces may trigger a biochemical cascade which comprises the activation of various types of periodontal ligament cells that could eventually favour inflammatory processes.Overall, evidence collected so far strongly supports the major role of SP in the development and maintenance of dental pain and inflammation. An increase in the expression and release of this molecule has been demonstrated following several pathological conditions and common dental procedures. Although physiological levels of SP are needed in order to guarantee correct blood flow in the dental pulp that can help tissue healing, from the available literature it clearly appears that high levels of the neuropeptide are associated with pain and persistent inflammation. Clinicians should be aware that most orthodontic procedures have an impact on the pulpodentine complex, which could generate several neurogenic and vascular reactions, including the release of SP. Although the number of reports in the literatures is only few, there is emerging the evidence that not all orthodontic procedures have a similar impact on SP release , 35, 37.A second approach to blunt the consequences of SP would be to target with appropriate pharmacological treatment either SP release or SP binding to its receptors present in the pulp. It has been documented that the injection of local anaesthetics results in the attenuation of SP release , 41. Thiad hoc studies are required to confirm or discard these hypotheses. Therefore, we believe that careful, evidence-based evaluation of the effects of single NSAIDs on the modulation of SP expression, neurogenic inflammation, and sensory neuron excitability in the dental pulp should be pursued in the future.To our knowledge, the effects on SP expression and release of common analgesic treatments used to control pain after dental procedures have not been intensely investigated to date. For instance, nimesulide reduces the concentration of pain mediators, including SP, in patients with knee osteoarthritis , but the


The purpose of this study was to design and to verify a new hearing-aid fitting strategy (Aescu HRL-1) based on the acoustic features of Mandarin. The subjective and objective outcomes were compared to those fitted with NAL-NL1  in Mandarin-speaking hearing-aid users. Fifteen subjects with sensorineural hearing loss participated in this preliminary study. Each subject wore a pair of four-channel hearing aids fitted with the Aescu HRL-1 and NAL-NL1 prescriptions alternatively for 1 month. Objective and subjective tests including the Mandarin Monosyllable Recognition Test (MMRT), Mandarin Hearing in Noise Test (MHINT), International Outcome Inventory for Hearing Aids (IOI-HA), and a sound-quality questionnaire were used to evaluate the performance of the two prescriptions. The mean MMRT scores were 79.9% and 81.1% for NAL-NL1 and Aescu HRL-1 respectively. They are not statistically different. The corresponding MHINT signal-to-noise ratios were 0.87 and 0.85 dB, also, no significant difference was found between these two strategies. However, in subjective questionnaires, overall, the sound-quality and IOI-HA scores were higher for Aescu HRL-1. The speech recognition performance based on Aescu HRL-1 is as good as that of NAL-NL1 for Mandarin-speaking hearing-aid users. Moreover, the subjects generally responded that Aescu HRL-1 provides a more natural, richer, and better sound quality than does NAL-NL1. Over the past 60 to 70 years, and especially during the most recent 35 years, there has been substantial research into the amplification needs of people with hearing impairments. Accordingly, many fitting strategies  for hearing aids have been developed to ensure that incoming sounds are audible whilst not being excessively loud for the hearing-aid users -3. So faThis situation led to the other prescription  being proposed in order to reduce the effect of upward spread of masking by presenting speech bands equally loud. Loudness equalization does not aim at normalizing frequency-specific loudness. Instead, the goal is to maximize speech intelligibility for every input level with the constraint that the overall loudness of speech must not exceed the normal level . The mos Approximately 1.2 billion people currently use Mandarin to communicate , but no Our laboratory has focused on the acoustic characteristics of Mandarin in recent years. Our results indicate that the frequency importance function of Mandarin is more weighted than English in the frequency band of 1000 to 6300 Hz, which has the summed importance of 72% in this band ; in addiThe main purpose of this study was to design a loudness normalization based fitting strategy according to the acoustic characteristic of Mandarin speech map. The strategy is called Aescu Hearing Research Lab – Version 1 (Aescu HRL-1). The second purpose was to verify this prescription and compared its objective and subjective outcomes with a commonly used loudness equalization strategy: NAL-NL1. Speech intelligibility was tested using objective tests, while sound quality was evaluated by a subjective questionnaire.The present study protocol was approved by Institutional Review Board on experimental ethics committee at National Taiwan University hospital (ID: 201003023R). All subjects provided written informed consent to participate.The Aescu HRL-1 fitting strategy is a nonlinear amplification method developed on the rationale of loudness normalization, and it is threshold-based. The difference in the loudness-growth curves between NH and HI individuals is used to set the gain of the hearing aid. The loudness-growth curve of NH subjects was derived by applying spline nonlinear interpolation to the equal-loudness contours of Suzuki et al. ,30. LoudPrevious studies showed that a fitting strategy based on loudness normalization may have some problems regarding the performance of speech intelligibility. In noisy environment, as both speech and background noise are dominated by energy in the low frequencies, the HI individuals could suffer from the effect of upward spread of masking, which compromises speech intelligibility . Our preIn addition, this modification decreases the gain for low input levels, and decreases the compression ratio when mapping to the WDRC architecture of hearing aids compared to the traditional methods of loudness normalization. van Buuren ,34 showeFifteen subjects  whose native language is Mandarin participated in this study. The subjects aged from 24 to 66 years with a mean of 48 years. They all have postlingual, bilateral and symmetric sensorineural hearing loss. The average pure-tone hearing thresholds  of both ears of each subject were all within the range 50–80 dB HL. The behind the ear (BTE) style hearing aids of FOCUS 4M, developed by Aescu in Taiwan, was used in this study. It is a 4-channel digital hearing aid and comprises eight filter bands and can provide independent compression in 4 channels. In addition, both the static and dynamic characteristics of the compression channels are adjustable, the bandwidth is from 200 to 5300 Hz, range of compression is from 1:1 to 1:5, and maximum output level is 129 dB SPL; additional features of FOCUS 4M includes directional microphone, noise reduction, feedback cancellation etc. FOCUS 4M can be set to the NAL-NL1 or Aescu-HRL1 prescriptions. The NAL-NL1 and Aescu HRL-1 fitting strategies were compared using different objective and subjective methods. The speech recognition abilities in quiet and noise were measured with the Mandarin Monosyllable Recognition Test (MMRT)  and the  The self-reported hearing instrument benefit and satisfaction measures were assessed using a self-designed sound-quality questionnaire  and the The study was conducted with single-blind design. MMRT and MHINT were administered to each subject to assess the unaided speech recognition ability before hearing aid fitting. Then, bilateral hearing aids were both fitted alternatively with NAL-NL1 and Aescu HRL1, the order was counterbalanced. After fitting with one prescription, the subjects had 3–4 weeks to acclimatize to the amplified sounds of the hearing aids provided by each fitting strategy. Then the subjects’ aided speech recognition ability was assessed again and they were asked to complete the sound quality and IOI-HA questionnaires. After the tests, the subjects were fitted using another fitting strategy and the same experimental procedures as for the first fitting were repeated.The Verifit hearing aid analyzer of Audioscan was used to evaluate whether the fitting is suitable or not based on speechmap. Speechmap creates a map of the amplified speech region within the residual auditory area using several simulated speech signals. Scollie and Seewald  providedWhen the LTASS curve of speechmap fits the targets of the fitting strategy at each frequency, it means a good fitting based on the speechmap method. t tests for comparisons: unaided versus NAL-NL1, p < 0.01; unaided versus Aescu HRL-1, p < 0.01; and NAL-NL1 versus Aescu HRL-1, p=0.601. There was no statistical significance between these two fitting strategies despite scores improving significantly after amplification.The correct rates for the speech recognition ability from the MMRT are shown in t tests for comparisons: unaided versus NAL-NL1, p<0.01; unaided versus Aescu HRL-1, p<0.01; and NAL-NL1 versus Aescu HRL-1, p=0.936. Again, no significant difference was found between these two strategies.The mean presentation level required for a 50% correct score in the MHINT in quiet for the 15 subjects see  was 43.3t tests for comparisons: unaided versus NAL-NL1, p=0.008; unaided versus Aescu HRL-1, p=0.003; and NAL-NL1 versus Aescu HRL-1, p=0.968. The signal-to-noise ratios required for a 50% correct rate in the MHINT in noise for the 15 subjects see  were 0.8p > 0.05).This study compared a modified loudness normalization prescription (Aescu HRL-1) with a loudness equalization prescription (NAL-NL1) implemented in a four-channel device, and applied to 15 subjects with flat and high-frequency hearing losses. The two prescriptions employed different amplification targets for these subjects. For example, for a moderate input level, the average gains prescribed by Aescu HRL-1 were 9.8, 0.5, and 0.3 dB higher at 250, 500, and 4000 Hz than those prescribed by NAL-NL1 across all 15 subjects. The trends of the target gains readily clarify the differences between Aescu HRL-1 and NAL-NL1, with the former based on the concept of loudness normalization amplification. The objective assessments revealed that the word intelligibility was significantly better for NAL-NL1 and Aescu HRL-1 than for the unaided condition, which means both fitting strategies improved the audibility of Mandarin words for HI individuals. However, the mean scores in the MMRT and MHINT did not differ significantly between the Aescu HRL-1 and NAL-NL1 fitting strategies, in other words, the Aescu HRL-1 can provide the same speech intelligibility for subjects NAL-NL1. Previous studies showed that the speech intelligibility was better for a loudness-equalization-based amplifier than for loudness normalization ,6. HowevIn addition to improve speech intelligibility, 88.4% of hearing-aid users seek improved sound quality . Hence, Hearing loss will affect the performance of children when learning to speak ,41. WhenIn summary, this study has demonstrated that the amplification characteristics of the two prescriptions rely on marked differences in overall gains, with the subjects showing only minor preferences to either prescription. However, if the resulting amplification characteristics from the two prescriptions differ substantially, then the subjects showed a predominant preference for Aescu HRL-1, whose prescription aims at provide a natural sound of high quality. A limitation of this study is that the experiments were conducted with a moderate speech level only, without the use of the other speech levels to evaluate the performance in objective measurement test, and did not consider the interactions between fitting strategy and the other hearing aid functions, such as speech enhancement, feedback cancellation, and directional microphone. Therefore, the overall effectiveness of the Aescu HRL-1 combined with these algorithms needs to be investigated in future studies.The results show that the speech intelligibility of the Aescu HRL-1 was as good as NAL-NL1 in objective tests. In subjective evaluations the subjects generally preferred Aescu HRL-1, responding that this prescription provided richer, smoother, and more natural sound compared to NAL-NL1. This pilot study shows the potential of this new hearing-aid fitting prescription in providing a sound quality similar to that of a prescription based on loudness normalization, and speech intelligibility similar to that of a prescription based on loudness equalization.Appendix S1The sound quality questionnaire.(PDF)Click here for additional data file.Appendix S2The international outcome inventory for hearing aids (IOI-HA) questionnaire.(PDF)Click here for additional data file.


Overall success of current tinnitus therapies is low, which may be due to the heterogeneity of tinnitus patients. Therefore, subclassification of tinnitus patients is expected to improve therapeutic allocation, which, in turn, is hoped to improve therapeutic success for the individual patient. The present study aims to define factors that differentially influence subjectively perceived tinnitus loudness and tinnitus-related distress.In a questionnaire-based cross-sectional survey, the data of 4705 individuals with tinnitus were analyzed. The self-report questionnaire contained items about subjective tinnitus loudness, type of onset, awareness and localization of the tinnitus, hearing impairment, chronic comorbidities, sleep quality, and psychometrically validated questionnaires addressing tinnitus-related distress, depressivity, anxiety, and somatic symptom severity. In a binary step-wise logistic regression model, we tested the predictive power of these variables on subjective tinnitus loudness and tinnitus-related distress.The present data contribute to the distinction between subjective tinnitus loudness and tinnitus-related distress. Whereas subjective loudness was associated with permanent awareness and binaural localization of the tinnitus, tinnitus-related distress was associated with depressivity, anxiety, and somatic symptom severity.Subjective tinnitus loudness and the potential presence of severe depressivity, anxiety, and somatic symptom severity should be assessed separately from tinnitus-related distress. If loud tinnitus is the major complaint together with mild or moderate tinnitus-related distress, therapies should focus on auditory perception. If levels of depressivity, anxiety or somatic symptom severity are severe, therapies and further diagnosis should focus on these symptoms at first. Subjective tinnitus is a sound that does not originate from an external or body sound source and that is heard only by those affected. Tinnitus is a widespread symptom with 30–40% of the adult population experiencing tinnitus during their life, and 0.5–2.5% being severely affected by a tinnitus that interferes with life quality Most therapeutic interventions focus on the reduction of tinnitus-related distress without primarily trying to reduce tinnitus loudness The subjectively perceived loudness of tinnitus can be recorded by numeric rating scales which typically range from 0 or 1 (low loudness) to 10 (high loudness) and which have been used in several studies on tinnitus e.g. . NumericAim of the present questionnaire-based study was to determine factors that differentially affect subjectively perceived tinnitus loudness and tinnitus-related distress. Data were collected from 4705 persons affected by tinnitus. The questionnaire included items about tinnitus characteristics such as localization and type of sound, tinnitus duration, subjectively perceived tinnitus loudness, and hearing impairment. Strength of tinnitus-related distress was recorded with the psychometrically validated short version of the Tinnitus Questionnaire MTQ, . DepressDuring September of 2010 a questionnaire with a total of 256 items was distributed by mail to all of the 13,349 registered patient members of the German Tinnitus Association . The questionnaire was accompanied by a letter in which the participants were informed, that by filling out and sending in the questionnaire they agreed to the use of their data for research purposes. 4752 questionnaires were received, and the data of 4705 questionnaires were entered into the data base. The rest was omitted mainly because of invalid membership numbers. The questionnaires were pseudonymized in that they contained the membership code but not the participants' names. The study protocol was approved by the local ethics committee (Ethikkommission II) of the Medical Faculty Mannheim of Heidelberg University and by the data safety commissioner of the Medical Faculty Mannheim of Heidelberg University according to the principles expressed in the Declaration of Helsinki.Besides information about age and gender the following information included in the questionnaire was used for the present study:In the questionnaire type of tinnitus sound, type of tinnitus onset (slowly/suddenly), its duration and localization as well as the time of daily tinnitus awareness was assessed. Subjectively perceived loudness was recorded on a numeric rating scale (T-NRS) from 0 (tinnitus audible only during silence) to 10 . To asses the potential presence of hearing impairments it was asked, if an audiogram was taken, if hearing impairment had been diagnosed by an otolaryngologist, and if hearing aids were used. Response options were ‘yes’ or ‘no’. This did not accurately reflect the amount of hearing loss, but did tell whether hearing impairments were uni- or bilateral and which side was affected.Tinnitus-related distress was addressed by the psychometrically validated brief version of the tinnitus questionnaire MTQ:  with sumThree psychometrically validated modules of the Patient Health Questionnaire (PHQ) were used to address depressivity (PHQ-9), anxiety (GAD-7), and somatic symptom severity (PHQ-15) Finally, one question each asked about difficulties to initiate and to maintain sleep, and the presence of chronic somatic morbidities as well as chronic pain and dizziness were recorded.Data management and data analysis were performed with the Statistical Package for the Social Sciences (SPSS) 18.0 for Windows  and SAS for Windows 9.2 . Percentages are reported for categorical variables % in  and meanTo identify the variables with the strongest association with MTQ and T-NRS respectively, groups with mild (MTQ-score≤7) versus severe (MTQ-score≥19) tinnitus-related distress, and with low (T-NRS≤2) versus high loudness (T-NRS≥8) were compared. Conceivably loudness and distress clusters do partially overlap. Data were categorized into major (problematic) versus minor with a cut-off score of ≥15 (major) for the variables PHQ-9, GAD-7 and PHQ-15 distinguishing severe levels of depressivity, anxiety and somatic symptom severity Data were derived from 4705 questionnaires. The age range was 18 to 94 years , and the overall female proportion was 40.9%. Mild tinnitus-related distress (MTQ-score≤7) was reported by 37.6%, whereas distress related to the tinnitus was judged as being intermediate (8≤MTQ-score≤18) by 49.0%, and 13.4% of the participants felt severely distressed by their tinnitus (MTQ-score≥19). Low subjective loudness was commonly associated with mild distress, whereas high subjective loudness tended to be associated with severe distress. These categories were not congruent, however. In 13 participants (0.3%) a subjective loudness score (T-NRS) of two and lower combined with severe tinnitus–related distress, whereas 209 participants (4.4%) reported a combination of loud (T-NRS≥8) but only mildly distressing tinnitus. Overall, the correlation between T-NRS and MTQ was found to be moderate (r = 0.524 Spearman's rho based on MTQ sum scores). Gender effects were not apparent, whereas older age and tinnitus duration of more than 5 years were associated with louder but not with a more distressing tinnitus. Sudden onset was prevalent, and of the 50% that named a possible cause, the majority (n = 1716) suspected stress followed by sudden hearing loss (1389) and noise trauma (354) as putative reasons. Ringing, continuous, binaural-central tinnitus prevailed, and unilateral tinnitus was more frequently located on the left ear which coincided with a higher incidence of left unilateral hearing impairment . The subAltogether 78.1% reported a hearing impairment that had been diagnosed audiometrically by an otolaryngologist. This percentage rose with age . Percentages of bilateral hearing impairment, binaural-central localization and permanent awareness of the tinnitus differed substantially when comparing low and high subjective tinnitus loudness .42% of the individuals indicating hearing impairments used hearing aids. 29.9% of them experienced a decrease while 5.1% experienced an increase in the subjective loudness of the tinnitus when using hearing aids. Participants with low subjective loudness (T-NRS≤2) as well as participants with mild distress (MTQ≤7) benefitted most from the use of hearing aids, whereas those with severe tinnitus-related distress (MTQ≥19) reported loudness increases most often and decreases least often .Correlations between depressivity, anxiety, somatic symptom severity and T-NRS were low . Correlations between MTQ and the PHQ-scales  exceeded those between MTQ and T-NRS (0.524). Most noteworthy was the high incidence of elevated PHQ scores in the group with severe tinnitus-related distress . CorrelaMore than 50% of the sample reported somatic comorbidities. Percentages were lower only in the group with low subjective loudness. Reports of sleep disturbances, chronic pain and dizziness peaked in the group with severe tinnitus-related distress. Analysis of OR revealed that sleep disturbances, chronic pain and dizziness were associated with tinnitus-related distress, and that all variables were associated with subjective loudness although to a lesser extent .Univariate analyses showed that groups with low and high subjective tinnitus loudness differed substantially (OR≥2 or ≤0.5) with respect to the percentage of subjects that was permanently aware of the tinnitus, somatic symptom severity, binaural/central localization of the tinnitus, anxiety and depressivity, binaural hearing impairment, tinnitus duration, and in the variables sleep problems, age, chronic pain, dizziness, and somatic comorbidities. Dominance of loudness over distress was most obvious for those with loud but only mildly distressing tinnitus (n = 209). In this group the low sum scores in the PHQ scales  were most notable.In contrast, groups with mild respectively severe tinnitus-related distress differed most in the percentage of elevated depressivity, anxiety, somatic symptom severity, and sleep disturbances. Permanent awareness and localization of the tinnitus as well as chronic pain and dizziness were less influential, whereas bilateral hearing impairment age and tinnitus duration had no substantial influence.The predominant characteristic in the group with severe tinnitus-related distress was the high prevalence of psychologically relevant symptoms as indicated by elevated scores in the PHQ scales. Conspicuous were also the high percentages of those with comorbid chronic pain and dizziness. Moreover, the majority had difficulties in initiating or maintaining sleep, and overall benefit from hearing aids was lower than average. Influence of the psychopathological variables was most obvious in a small cluster of 13 subjects who reported low subjective tinnitus loudness but severe tinnitus-related distress. Mean sum scores of PHQ-9 (13.3 [5.0]), GAD-7 [12.2 [6.5]) and PHQ-15  were less frequent than average.In a step-wise regression analysis the predictive power of auditory and non-auditory variables on tinnitus-related distress respectively subjective tinnitus loudness with the factors showing OR of 2 and above or of 0.5 and below was calculated. Factors found to be relevant for subjective tinnitus loudness was above all the factor “permanent awareness” of the tinnitus followed by “binaural/central localization”, sleep problems and pain. In contrast, most influential variables on tinnitus-related distress were depressivity and anxiety followed by sleep problems and permanent awareness of the tinnitus. In addition the variables binaural/central tinnitus localization as well as somatic symptom severity and pain had significant influence . NoteworThe results of the present study are based on data obtained from 4705 participants with tinnitus. The observed distribution of tinnitus characteristics is in accordance with those found in epidemiological tinnitus studies To our knowledge this study analyzes the largest sample of subjects with tinnitus that has ever been evaluated with psychometrically validated questionnaires addressing depressivity, anxiety and somatic symptom severity in conjunction with subjective tinnitus loudness and tinnitus-related distress. The observed percentages of severe depressivity and anxiety are slightly higher than in the general population In addition, severe somatic symptom severity is increased in the group with severe tinnitus-related distress compared to the study population in general. The threshold we used for the distinction between moderate and severe cases requires the presence of at least seven bothering somatic symptoms, and is seen as a reliable distinction between presence of somatoform disorders in comparison to their absence In agreement with former tinnitus studies An important issue that cannot be settled with this cross-sectional survey is whether these comorbidities are primary or secondary to the tinnitus. Longitudinal studies with a small number of acute tinnitus patients suggest that psychopathological conditions exist beforehand and constitute risk factors for the development of a distressing tinnitus or that they arise together with the tinnitus The rate of hearing impairment in the present study is high. Its incidence increases with age, and bilateral hearing impairment is more frequent in the group that experiences loud tinnitus. These findings agree with those of others Hearing impairment is thought to be the permissive condition for the development of the tinnitus perception. Therefore restoring auditory input is expected to reduce the subjectively perceived tinnitus loudness. Though, results of such interventions are variable and the overall success rate is low Some limitations apply to the present analysis. Our tinnitus population may not represent the tinnitus population as a whole, but may be dominated by individuals that are concerned by their tinnitus and became active by joining a patient organization. Therefore the reported characteristics may not be entirely representative for the general tinnitus population. Furthermore the evidence relies on self-report questionnaires and therefore may be influenced by misconceptions. However, characteristics of the investigated tinnitus population are in line with the published literature Subjectively perceived tinnitus loudness as measured here represents a distinctive quality of tinnitus which needs to be assessed separately and in addition to tinnitus-related distress and to psychoacoustic tinnitus characteristics. This can be done effectively by numeric rating scales. Study participants with a severely distressing tinnitus expressed elevated levels of depressivity, anxiety, and somatic symptom severity, and the high incidence of sleep problems, chronic pain and dizziness in the highly distressed tinnitus patients appears to be associated foremost with these factors. Therefore, especially in subjects with high tinnitus-related distress, the potential presence of severe depressivity, anxiety and somatic symptom severity should be assessed separately from tinnitus-related distress by validated psychopathology questionnaires.A combination of the MTQ questionnaire with established questionnaires addressing psychopathologies such as PHQ-9, GAD-7, and PHQ-15 in conjunction with audiological examination and recording of the subjectively perceived tinnitus loudness represents a powerful and easy to handle tool to characterize tinnitus patients. While these parameters are already assessed in specialized tinnitus centres, they also need to be evaluated during routine otolaryngologic examination of tinnitus patients. As suggested by the differential effect of hearing aids in distressed and non-distressed participants, a comprehensive characterization may optimize patient allocation and consequently the therapeutic outcome of existing tinnitus therapies.