Recent studies on animal models have shown that noise exposure that does not lead to permanent threshold shift (PTS) can cause considerable damage around the synapses between inner hair cells (IHCs) and type-I afferent auditory nerve fibers (ANFs). Disruption of these synapses not only disables the innervated ANFs but also results in the slow degeneration of spiral ganglion neurons if the synapses are not reestablished. Such a loss of ANFs should result in signal coding deficits, which are exacerbated by the bias of the damage toward synapses connecting low-spontaneous-rate (SR) ANFs, which are known to be vital for signal coding in noisy background. As there is no PTS, these functional deficits cannot be detected using routine audiological evaluations and may be unknown to subjects who have them. Such functional deficits in hearing without changes in sensitivity are generally called “noise-induced hidden hearing loss (NIHHL).” Here, we provide a brief review to address several critical issues related to NIHHL: (1) the mechanism of noise induced synaptic damage, (2) reversibility of the synaptic damage, (3) the functional deficits as the nature of NIHHL in animal studies, (4) evidence of NIHHL in human subjects, and (5) peripheral and central contribution of NIHHL. Noise-induced hidden hearing loss (NIHHL) refers to any functional impairment seen in subjects with noise exposing history but no permanent threshold shift (PTS). This is different from the conventional definition of noise-induced hearing loss (NIHL), which is based on changes in auditory sensitivity or threshold shift . TherefoPhysiologically, variations in auditory sensitivity following exposure to noise are largely due to the functional status of outer hair cells (OHCs) in the cochlea, which provide mechanical amplification of soft sounds , 3. NoisAlthough some early reports claimed that reversible noise-induced IHC pathologies were responsible for TTS , 16, IHCNIHHL first manifests as reduced output of the auditory nerve at high sound levels, without affecting the hearing threshold. This reduction has been found in both animals , 20–23 aAs NIHHL is initiated at the synapse between the IHCs and SGNs, which silences the auditory nerve fibers (ANFs) that extend from them, the corresponding disorder is categorized as a cochlear neuropathy  , 26. PreAccumulated evidence has shown that the synapses between IHCs and type-I SGNs are sensitive to noise and the damage to this synapse is likely to be the bases for NIHHL. The synapse is characterized by presynaptic dense bodies termed ribbons –35, whicNoise exposure causes damage to both the presynaptic ribbons and postsynaptic nerve terminals of the ribbon synapses , 46–48. 2+ signaling involving CaV-channels, presynaptic Ca2+ levels and storage, and guanylate cyclase-activating protein-2 . IntereThere is some debate about whether noise-induced ribbon synapse damage is reversible. The first quantitative study of noise-induced ribbon synapse damage in CBA mice  reportedOur studies on guinea pigs have revealed a clear recovery in the synapse count following a massive initial loss induced by noise exposure that did not lead to PTS , 23. AltIn a recent review, it was argued that the recovery of CtBP2/PSD counts in guinea pig cochleae following noise exposure reported in our studies may be attributable to up/downregulation of the synaptic protein rather than regeneration of synaptic connections . HoweverRibbon synapses exhibit spatial differences around IHCs; that is, the synapses at the modiolar side of an IHC have relatively small ribbons but larger postsynaptic terminals, whereas those at the pillar side have relatively large ribbons but smaller terminals . This spIn NIHHL, low-SR ANFs appear to be more vulnerable to noise than high-SR units. Selective loss of low-SR ANFs has been found following exposure to noise that did not lead to PTS . PresumaIntensity coding in the cochlea is defined as the ability of ANFs to encode the sound intensity or the change of sound intensity. This ability is determined primarily by the spike rate (or the change of spike rate) of individual ANF in response to sound intensity change and the number of functional ANFs. Therefore, the intensity coding deficits can be evaluated in both evoked field potential and single-unit recordings. Deficits in intensity coding were first suggested by a reduction in wave I of the auditory brainstem response (ABR) , 49, as The deterioration in intensity coding following no-PTS noise exposure was manifested as a reduction in the driven spike rates  of ANF units that were tested only at one sound level . Such chTemporal processing ability in the cochlea as well as in the whole auditory pathway is defined as the ability to follow the quick change of acoustic signals. In human subjects, the process involves both bottom-up and top-down mechanisms; but in animal models, only bottom-up mechanisms are tested (see reviewed by ). Many dPhase locking is a mechanism for the auditory coding of temporal envelopes. A temporal deficit in phase-locking responses has been proposed based on selective loss of low-SR units and the functional features of this group of ANFs , 26, 69,So far, there appear to be two models for the development of coding deficits in NIHHL. One model suggests that the coding deficit or synaptopathy is simply due to the loss of low-SR ANFs. Since those units have unique functions in signal coding, the loss of those functions is predicted as the consequences. Evidence from our own laboratory suggests another model. That is, the coding deficits are developed as the result of unhealthy synaptic repair after initial disruption. We found that the noise-induced synaptic damage in guinea pigs under NIHHL is largely repairable, leaving only a small amount of synapses not being reestablished. Therefore, the coding deficits or synaptopathy cannot be simply attributed to the loss of SR units. Since the coding deficits are seen at the time when the synapse counts are largely recovered, we believe that the coding deficits likely occur in the repaired synapses (most of them innervating low-SR ANFs). Studies are needed to verify which model is more likely the case in human subjects.Hearing loss impacts auditory perception. It has long been recognized that subjects with normal audiograms may have perceptual difficulties, and this is especially true in the elderly. Age-related hearing loss with threshold elevation is termed peripheral presbycusis, whereas the perceptual difficulties seen in the elderly without threshold shift are usually termed central presbycusis . For exaBased on recent progress in functional deficits in cochlear coding, such separation between peripheral and central presbycusis is likely to be incorrect. The so-called central presbycusis may, at least in part, result from disorders in the auditory periphery. The coding deficits related to the loss of low-SR ANFs had been described as a type of auditory neuropathy and/or synaptopathy even before any of the predicted deficits were identified. Data on changes in the SR distributions of ANFs suggest the reestablishment of synapses following an initial disruption that was selective to low-SR units . AlthougIt should be noted that there is now a tendency in the literature to consider NIHHL to be a purely peripheral issue, a result of the overcorrection of the “central presbycusis.” However, despite the strong evidence for a peripheral contribution, the central contribution to the problems seen in NIHHL should not be neglected. In other words, it may be more constructive to assume that there are both peripheral and central contributions to NIHHL. It is well known that hearing loss (with elevated threshold) can induce central changes, which can result in deteriorations in signal processing. Studies aiming to distinguish the role of central plasticity from that of ribbon synapse damage are rare. One such report found that an increase in central gain was responsible for tinnitus in human subjects with typical damage seen in NIHHL ) but normal hearing threshold . In an eOne of the central impacts of hearing loss due to damage to peripheral auditory organ is imbalance between excitation and inhibition, resulting in hyperactivity and/or hyperresponsiveness in the central auditory system (see reviews in –80). TheIn a brief summary, we use Although more studies on the impact of noise on human hearing showing no changes in auditory sensitivity are required, evidence suggesting the occurrence of NIHHL in human subjects is being accumulated. This is supported by thorough research on the signal perception deficits experienced by subjects with a history of noise exposure but normal thresholds . Since tThe clinical implications of NIHHL are manifested by the fact that noise exposure causing NIHHL occurs frequently in daily life and impacts much more general population . Such noIn future, the coding deficits and related synaptic repair in NIHHL should be further investigated in a laboratory setting. Since the ribbon synapse is the first gating point for temporal processing in auditory pathway, the observed coding deficits suggest a clear peripheral origin for the decline in temporal processing and perceptual difficulties during aging. Whether and how the synaptic damage will impact the central auditory processing need to be investigated in a manner that is clearly differentiated from the impact of hearing threshold shift. Moreover, the coding function of ANFs should be observed over a long period of time following exposure to noise to determine whether the coding deficits are temporary or persistent. We are currently collecting data using electron microscopy, as well as conducting an analysis on the potential changes of the molecular structures of ribbons and PSDs, in an attempt to elucidate the morphological/molecular mechanisms responsible for functional changes of repaired ribbon synapses. It would also be interesting to understand the reasons for the extreme sensitivity of low-SR synapses to noise, as well as elucidate possible methods to prevent damage. Laboratory studies should also aim to explore the mechanisms of synaptic repair in the cochlea, as well as reveal the factors that influence repair in order to promote it.To translate the knowledge to clinic, investigation is needed to establish good measures for detecting NIHHL in human subjects. Although ABR wave I is useful for evaluating synaptopathy caused by noise that does not induce PTS, its reliability and sensitivity are questionable in human subjects where the ABR amplitude is small, and other methods should be explored. A very recent report suggests the use of ABR latency as the marker of NIHHL . The stuTo date, the most promising methods for diagnosing cochlear synaptopathy are related to selective loss of low-SR ANFs, the subcortical steady state responses (SSSR) , 94. Bas


Streptomyces griseoviridis NRRL 2427, are a pair of synergistic streptogramin antibiotics and very important in treating infections of many multi-drug resistant microorganisms. Three transporter genes, sgvT1–T3 have been discovered within the 105 kb GV/VG BGC, but the function of these efflux transporters have not been identified.Griseoviridin (GV) and viridogrisein , both biosynthesized by a distinct 105 kb biosynthetic gene cluster (BGC) in sgvT1–T3 expression. Above all, overexpression of sgvT1 and sgvT2 enhances transmembrane transport leading to steady production of GV/VG in titers ≈ 3-fold greater than seen for the wild-type producer and without any notable disturbances to GV/VG biosynthetic gene expression or antibiotic control.In the present study, we have identified the different roles of these three transporters, SgvT1, SgvT2 and SgvT3. SgvT1 is a major facilitator superfamily (MFS) transporter whereas SgvT2 appears to serve as the sole ATP-binding cassette (ABC) transporter within the GV/VG BGC. Both proteins are necessary for efficient GV/VG biosynthesis although SgvT1 plays an especially critical role by averting undesired intracellular GV/VG accumulation during biosynthesis. SgvT3 is an alternative MFS-based transporter that appears to serve as a compensatory transporter in GV/VG biosynthesis. We also have identified the γ-butyrolactone (GBL) signaling pathway as a central regulator of Our results shows that SgvT1–T2 are essential and useful in GV/VG biosynthesis and our effort highlight a new and effective strategy by which to better exploit streptogramin-based natural products of which GV and VG are prime examples with clinical potential.The online version of this article (doi:10.1186/s12934-017-0792-8) contains supplementary material, which is available to authorized users. Streptomyces griseoviridis NRRL 2427 generates two types of unrelated streptogramins; these include griseoviridin (GV) belonging to the A-type cyclic polyunsaturated macrolactone subclass, and viridogrisein (VG), a B-type cyclic depsipeptide counterpart to GV also referred to as etamycin  and ATP-binding cassette (ABC) families and constitute an essential self-resistance mechanism to efficiently secrete antibiotics as they are constructed thereby protecting the producing microbe from the effects of its own secondary metabolism –10. ThisStreptomyces . The. ThesgvTendulae) , 23–25. endulae) . SgvT3 csgvT2 gene represents the point of differentiation between upstream VG biosynthesis and downstream GV biosynthesis components and encodes the only 551 aa ABC transporter , DrrA (S. peucetius), KasK (S. kasugaensis), MtrA (S. argillaceus), OleC (S. antibioticus) and PdmR1 (Actinomadura hibisca) . The ΔsgvT2 strain also suffered sharply reduced GV/VG production efficiencies with yields only on par with 30–35% of that observed with WT producer; metabolite titers for the ΔsgvT2 were 10.05 ± 0.09 μg/mL GV and 11.21 ± 0.35 μg/mL VG after fermentation for 108 h   were all found to express sgvT1–T3 at rates similar to that seen with the WT producer -type “quorum sensing” signal molecules of pathway-specific activation of GV/VG biosynthesis has been previously shown . Inactivery Fig. .Fig. 5PrsgvT1 and ΔsgvT2 mutant strains both produce GV and VG with comparable efficiencies up to approx. 48 h. However, differences in biosynthetic efficiencies between the two strains become noticeable starting at about 48 h and continuing to 120 h where the difference is most pronounced , sgvR1 (repressor gene), sgvR2, R3 (activator genes), GV/VG backbones biosynthetic genes sgvE1/sgvD1 (GV/VG backbone biosynthetic genes), and sgvT1–T3 [sgvT2 correlated very closely with those found for WT strain at during the early (up to 60 h) phases of GV/VG biosynthesis. This was the case also for the intermediate (120 h) stage of biosynthesis presumably because intact SgvT1 and SgvT3 could ensure GV/VG transmembrane transport needed to prevent intracellular accumulation of GV and/or VG . Placement under the control of the ermE promoter afforded approximately a 20-fold over-expression of sgvT1 and a 19-fold over-expression of sgvT2. As expected, the high-yielding WT::sgvT1–T2 strain was found to generate GV/VG in substantially greater yield than does the native S. griseoviridis. Commensurate with expectation, GV and VG yields relative to the WT producer increased by ≈ 3-fold [(106.85 ± 1.81 μg/mL GV/106.61 ± 1.12 μg/mL VG) see Fig. sgvT1–T2 were achieved even in the absence of antibiotic generally used to maintain selective pressure  and cefT  also failed to impact natural product titers [Transporter-encoding genes within secondary metabolite biosynthesis gene clusters are central to cell viability and homeostasis; both rely heavily on a cell’s ability to pump antibiotics and other toxic metabolites out of the cell –14. Advat titers , 31. TheS. griseoviridis producer also supports the notion that effective efflux systems may require two dramatically different types of transporter proteins to work synergistically to maintain homeostasis within the producer.SgvT1 and SgvT2 are both necessary transporters involved in GV/VG biosynthesis; inactivation of either protein significantly decreased GV/VG fermentation titers. SgvT1 is a rare 15 TMS-containing MFS transporter. Alternatively, SgvT2 is composed of two NBD domains but is devoid of any TMS. The absence of TMS elements in SgvT2 suggests a reliance upon other TBD domain-containing transporters in order for SgvT2 to be involved in any kind of effective GV/VG efflux system. That two distinct chemical species (GV and VG) are generated by the sgvT1 and ΔsgvT2 mutant strains shed tremendous insight into the roles of these transporter genes. These analyses make clear that SgvT1 is pivotal to metabolite export and that its inactivation is only minimally compensated for by either SgvT2/T3 or perhaps other transporters within the producer genome. This is in stark contrast to the case of sgvT3 inactivation, which, based on our results, is readily compensated for by an intact SgvT1/T2 transport couple.Griseoviridin and VG are both excellent examples of streptogramins and previous work has placed a great deal of importance on understanding mechanisms of streptogramin cellular export during biosynthesis –10. We fsgvT1–T3 do not appear to directly affect the expression of GV/VG biosynthetic or activating genes. Only the ΔsgvT1 mutant appeared to have any of these gene’s expression indirectly inhibited, presumably in response to intracellular environmental adaptations. Moreover, we found that sgvT1–T3 are regulated by the GBL signaling pathway, which appears to synchronize with GV/VG biosynthesis without directly influencing GV/VG titers. On the basis of these findings we envision that, when the S. griseoviridis (WT or mutants) producer receives the GBL signal to activate GV/VG biosynthesis, sgvT1–T3 expression ensues to coordinate prompt GV/VG secretion thereby averting intracellular GV/VG accumulation. In this way, we posit that GBL signaling plays a key role in GV/VG biosynthesis by maintaining cellular homeostasis and coordinating GV/VG biosynthesis and transmembrane export -type signaling, which appears to synchronize with GV/VG biosynthesis and avert excessive intracellular GV/VG accumulation. Enabled by the discovery and application of two critical transporters SgvT1/T2, we constructed a new steady GV/VG high-producing strain in titers about threefold greater than seen for the wild-type producer and without any notable disturbances to GV/VG biosynthetic gene expression or environmental antibiotic control. Consequently, our effort highlighted a new strategy to better exploit streptogramin-based natural products and demonstrate that enhancing self-resistance mechanisms in antimicrobial producing organisms is a valuable approach to safely and efficiently improving metabolite production processes.S. griseoviridis NRRL 2427 and related mutant strains were executed as previous described [The general methods and materials used, as well as, all bacterial strains, plasmids, and culture conditions have all been previously described , 2. Fermescribed , 2.sgvT1–T3 in S. griseoviridis NRRL 2427 to construct the corresponding three mutants ΔsgvT1–T3  was constructed by two steps. The first step entailed introduction of sgvT1 by conjugation with pPWW50Apr-sgvT1 and screening via apramycin resistance as previous described [sgvT2 gene was then introduced by conjugation to pSET152AKE-sgvT2 and screening for desired fusions was carried out by kanamycin resistance screening as previous described [The escribed . The sgvS. griseoviridis NRRL 2427 WT strains, two mutants ΔsgvT1/T2 and the WT::sgvT1–T2, were fermented and subsequent metabolite analyses carried out as previously described [Four strains, the g, 10 min) to separate precipitates and cells from the supernatant [g for 15 min; both the supernatants and pellet contents were then subjected to HPLC analysis.To measure GV/VG production averages included in the confidence interval, two standard curves were generated using analytically pure sample and HPLC analysis  and total RNA was extracted from the frozen pellet using the SV total RNA purification Kit (Promega). RNA samples were subjected to Dnase I (Promega) digestion according to the manufacturer instructions. First-strand cDNA synthesis was accomplished using Invitrogen’s SuperScript™ Kit and second step PCR was carried out under previously indicated conditions [p < 0.05 and p < 0.01) was used to evaluate the significance of the difference between each sample.Mycelia harvested at specific time points were powderized using in Nnditions . Control


Eptesicus fuscus, during the period in which echolocation vocalizations increase dramatically in frequency. We found that seven genes were significantly upregulated in juveniles relative to adults, and that the expression of four genes through development correlated with estimated age. Compared to available data for mice, it appears that expression of some hearing genes is extended in juvenile bats. These results are consistent with a prolonged growth period required to develop larger cochlea relative to body size, a later maturation of high frequency hearing, and a greater dependence on high frequency hearing in echolocating bats.For echolocating bats, hearing is essential for survival. Specializations for detecting and processing high frequency sounds are apparent throughout their auditory systems. Recent studies on echolocating mammals have reported evidence of parallel evolution in some hearing-related genes in which distantly related groups of echolocating animals , cluster together in gene trees due to apparent amino acid convergence. However, molecular adaptations can occur not only in coding sequences, but also in the regulation of gene expression. The aim of this study was to examine the expression of hearing-related genes in the inner ear of developing big brown bats,  Echolocating bats have among the highest frequency hearing in the animal kingdom . While hTmc1) and Prestin/SLC26A5, exhibit convergence between the two distantly related groups of echolocating bats, or even between echolocating bats and whales, such that gene trees sometimes group echolocators together to the exclusion of non-echolocators [The importance of hearing to echolocators has been illustrated by a number of recent studies examining the molecular evolution of genes involved in hearing in bats. Several genes known from human and mouse studies to be crucial for normal hearing, such as transmembrane channel-like 1 (locators –17. Whillocators  and develocators .Eptesicus fuscus) is an insectivore that hunts in edge spaces between open and cluttered environments [The big brown bat .Because of their dependence on hearing for survival and their relatively well-developed auditory systems, echolocating bats provide a valuable opportunity to examine postnatal hearing development in an auditory specialist. Laryngeally echolocating bats possess larger cochlea  relativeEptesicus fuscus were captured in the wild under a permit from the Maryland Department of Natural Resources. All twelve juvenile subjects were born in captivity. Because they were group-housed and cluster together, exact dates of birth could not be directly recorded. Instead, forearm length was measured with calipers and used to estimate age [Guide for the Care and Use of Laboratory Animals, and were approved by the Johns Hopkins University Institutional Animal Care and Use Committee (protocol BA14A111). Samples were also obtained from two adult individuals under a protocol approved by the University of Maryland Institutional Animal Care and Use Committee (R-13-76).Pregnant female mate age . Forearmmate age . EstimatInner ear samples, consisting of the entire otic capsule (both cochleae and vestibular organs), were collected immediately post-mortem and placed into liquid nitrogen prior to storage at -80°C until extraction. Both left and right cochleae from an individual were pooled and processed together. Samples were homogenized with a mortar and pestle while submerged in liquid nitrogen. RNA extraction was performed using a mirVana kit (Ambion), with added proteinase K (Sigma Aldrich) to improve yield . All samEptesicus fuscus and all bats of the genus Myotis , were downloaded from GenBank (NCBI) and aligned using Clustal Omega (EMBL-EBI). Sequences from Myotis spp. were included in order to reduce the risk of designing primers in regions with polymorphic sites. All primer pairs were designed within the same exon to permit preliminary testing on genomic DNA.Candidate genes were selected based on one or more of the following criteria: upregulated in an echolocating bat vs. a non-echolocating bat ; upreguEptesicus fuscus transcript, exonic regions of the Myotis lucifugus transcript, as identified in Ensembl, were blasted against the transcript for Eptesicus fuscus. If the Myotis transcript was not available in Ensembl, the mouse (Mus musculus) transcript was used instead. If the exonic region was conserved among Eptesicus and Myotis spp., it was entered into Primer-BLAST (NCBI). Potential primer pairs were checked for specificity against Eptesicus fuscus RefSeq data, potential for cross- and self-dimerization, and potential to form hairpins using Beacon Designer (Premier Biosoft). Only primers that were 100% conserved across all known transcripts from Eptesicus and Myotis spp. were used for quantitative PCR. Primer sequences are given in To identify exons in an Five-point dilution series (1:3 or 1:4) were performed for each gene and only primer pairs with efficiencies greater than 90% after exclusion of non-linear dilutions  were selected for use. Post-amplification melt curves were checked to ensure each product consisted of a single, narrow peak, and gel electrophoresis was performed for each amplicon to ensure a single product of correct size was produced during amplification.GAPDH) was included as a reference gene on each 96-well plate. Fluorescence was measured using a Roche 480 Lightcycler and melt curves were measured immediately after the completion of all amplification cycles. Technical replicates that reached threshold two or more cycles earlier or later than the other two replicates were excluded from analyses.For each primer pair, 20 μL reactions were prepared for each of the samples in triplicate using SYBR Select Master Mix (Thermo Fisher). Glyceraldehyde-3-phosphate dehydrogenase . Fold expression was calculated as the efficiency-adjusted amplification factor raised to the negative delta-delta CT. Average CT and calculated fold expression values are given in For each sample-primer combination on a given plate, the comparative CT method  was usedWe performed t-tests to determine whether the mean adjusted fold expression values of juveniles differed from adults for 13 genes. We also fitted least squares regression lines between estimated age and adjusted fold change to identify genes that exhibited age-dependent expression. All statistical analyses were performed in JMP 13.0.0 (SAS Institute). Figures were generated in JMP and MATLAB R2015a (The Mathworks).Bmp7), carcinoembryonic antigen-related cell adhesion molecule 16 (Ceacam16), collagen type XI alpha 2 chain (Col11A2), POU class 4 transcription factor 3 (Pou4f3), transmembrane channel-like 2 (Tmc2), and USH1 protein network component harmonin (Ush1C), and higher in juveniles for the remaining two genes—gap junction protein beta 2 (Gjb2) and POU class 3 transcription factor 4 (Pou3f4).Of the 13 genes tested, eight exhibited differential expression between juveniles and adults ; Fig 1. Pou3f4), transmembrane channel-like 1 (Tmc1), and gap junction protein beta 2 (Gjb2) and 6 , and their deletion disrupts TM structure [Tectb improved the frequency selectivity of the BM and neural response at high frequencies [Ceacam16 may stabilize interactions between TM glycoproteins, such that cochlear amplification becomes unstable without it [Col11A2 and Ceacam16 may, therefore, result in a TM structure which allows bat hair cells to effectively amplify high frequency sounds.Both tructure , 56, restructure , 54. Thetructure . Reducinquencies . Ceacam1thout it . The uprPou4f3 showed the greatest difference in expression between age groups  compared to a non-echolocating bat (Cynopterus sphinx) [Gjb2 appears critical for cochlear function and is implicated in the most common form of congenital deafness in humans [Gjb6 has also been linked to human deafness [Gjb6 knockdown in mice are less severe than those of Gjb2 and may be partly caused by associated downregulation of Gjb2 [Four genes were significantly upregulated with age in juvenile bats. Of these, complete  and 2. Tple size . While l cochlea , we foun sphinx) . Gjb2 apn humans , 87. Gjbdeafness , althoug of Gjb2 , 88.Gjb2 and Gjb6 may reflect greater numbers of gap junctions in the bat cochlea. Both genes may participate in the recycling of potassium, the major charge carrier in transduction . Condit hearing , consist hearing –93. The  hearing , 34. A r hearing .Gjb2 and Gjb6 upregulation may provide some protection against hearing loss in echolocating bats, which depend on hearing throughout their long lives. Conditional knockdown of Gjb2 in mice at P18 resulted in greater susceptibility to noise-induced hearing loss at P30 and P45 [Gjb6 exhibited abnormal epithelial repair after hair cell loss and reduced intercellular communication between supporting cells [Gjb2 and Gjb6 expression during juvenile development in bats may, therefore, be associated with a system of gap junctions that facilitates cochlear protection or repair. After an hour of broadband noise exposure at 152 dB SPL, adult big brown bats showed no significant threshold shifts [ and P45 , and micng cells . Cx26 anng cells . The incd shifts , 98, incd shifts . Additiod shifts , and it Pou3f4 is a transcription factor that has been implicated in X-linked non-syndromic deafness [Pou3f4 mouse mutants exhibit audiological and balance impairments, reduced coiling of the cochlea [Pou3f4 from otic mesenchyme causes defasciculation of spiral ganglion neurons [Pou3f4 in the developing bat inner ear may be linked to cochlear elongation and functional organization. One report did not find evidence of positive selection on Pou3f4 among echolocating bats [deafness . Pou3f4  cochlea , and def cochlea . Deletio neurons , which c neurons . These sing bats , suggestTmc1 encodes a MET channel protein [Tmc1 expression in the inner ear of mice from P9 to P19, with a net decrease of approximately 8% over the period [Tmc1 we observed in developing big brown bats is consistent with the latter study, and with a transcriptomic comparison of the inner ears of bats which showed that 18 hearing-related genes were upregulated in an echolocating bat compared to a non-echolocating bat, including Tmc1, which was also upregulated in echolocating bats relative to mice and rats [ protein  that loc protein  and whic protein . Reportse period . Anothere period . The incand rats .Tmc1 could reflect a greater number of MET channels per hair cell, which might increase sensitivity to high frequencies by strengthening the influx of calcium and reducing the adaptation time of hair cells (reviewed in [Porichthys notatus), fluctuations in the expression of a calcium-activated potassium (BK) channel conferred greater hearing sensitivity during the breeding season [Tmc1 had faster adaptation times than those expressing only Tmc2 or only a Tmc1 mutant [Although the nature of our samples (entire inner ears) did not permit examination of gene expression specifically in the cochlea or its basal, high frequency region, the upregulation of iewed in ). In midg season , and knog season . Alterna1 mutant , MET chaTmc1 and Gjb2 mouse mutants exhibit hearing loss without vestibular dysfunction, illustrating their greater importance for audition [Tmc1 exhibits both sequence convergence [Although only a small set of genes were examined in this study, and we did not manipulate gene expression directly and monitor subsequent phenotypic effects, this study provides the first insight into the developmental expression of hearing genes in echolocating animals. Without separation of the cochlea from the vestibular organs, it is not possible to ascribe expression differences to one section of the inner ear or the other. However, audition , 60. Furaudition –17, as hvergence  and uprevergence , this reS1 Table(XLSX)Click here for additional data file.


Despite great advances in hearing-aid technology, users still experience problems with noise in windy environments. The potential benefits of using a deep recurrent neural network (RNN) for reducing wind noise were assessed. The RNN was trained using recordings of the output of the two microphones of a behind-the-ear hearing aid in response to male and female speech at various azimuths in the presence of noise produced by wind from various azimuths with a velocity of 3 m/s, using the “clean” speech as a reference. A paired-comparison procedure was used to compare all possible combinations of three conditions for subjective intelligibility and for sound quality or comfort. The conditions were unprocessed noisy speech, noisy speech processed using the RNN, and noisy speech that was high-pass filtered . Eighteen native English-speaking participants were tested, nine with normal hearing and nine with mild-to-moderate hearing impairment. Frequency-dependent linear amplification was provided for the latter. Processing using the RNN was significantly preferred over no processing by both subject groups for both subjective intelligibility and sound quality, although the magnitude of the preferences was small. High-pass filtering (HPF) was not significantly preferred over no processing. Although RNN was significantly preferred over HPF only for sound quality for the hearing-impaired participants, for the results as a whole, there was a preference for RNN over HPF. Overall, the results suggest that reduction of wind noise using an RNN is possible and might have beneficial effects when used in hearing aids. Airflow around a hearing aid can lead to turbulence at the microphone ports. The resulting random fluctuations in air pressure are often referred to as wind noise . This noWind noise varies with wind angle, microphone location, and across and within styles of hearing aids . For modAmelioration of the effects of wind noise in hearing aids is based on two approaches: acoustic design modifications and signal processing . The firThe second approach uses digital signal processing techniques to determine whether wind noise is present and to reduce wind noise when it is detected. Detection of wind noise is usually based on the fact that it is largely uncorrelated at the two (or more) microphones of a hearing aid, despite the microphones being located close to one another , whereasIn the past few years, Machine Learning (ML) has been widely used to achieve substantial improvements in computational tasks in the visual, auditory, speech, and language domains. A major advance came from the use of artificial neural networks with more than two layers . These cSeveral ML-based approaches using DNNs to segregate speech from masking noise have been shown to improve speech perception in noise for normal-hearing and hearing-impaired listeners and users of cochlear implants . RecentlThis article reports a study of wind noise reduction using an ML algorithm based on a deep (multilayer) LSTM model. For brevity, this is referred to hereafter simply as the RNN. The RNN was first trained to predict the ideal ratio mask , using recordings of speech corrupted by wind noise. The clean (noise-free) speech was used to estimate the target IRM. Then, the trained model was used to process the noisy speech, so as to attenuate time-frequency segments with low speech-to-noise ratio (SNR) while retaining segments with high SNR. The effects of the algorithm on judged speech intelligibility and sound quality were assessed for speech signals affected by wind noise, recorded using the microphones of behind-the-ear (BTE) hearing aids. Simple high-pass filtering was used for a comparison condition.The study was approved by the Psychology Research Ethics Committee of the University of Cambridge and was conducted in accordance with the Declaration of Helsinki.Eighteen native English-speaking participants took part in the experiment. Audiometric thresholds were measured for audiometric frequencies from 0.25 to 8 kHz, using a Grason-Stadler GSI-61 audiometer. Nine of the participants had normal hearing , with audiometric thresholds lower than 20 dB HL for all measured frequencies, and nine had hearing loss. Only the better-hearing ear of each participant was tested (based on the average threshold across 0.5 to 4 kHz). The sex, age, and audiometric thresholds for the test ear of the hearing-impaired participants are shown in The wind and speech azimuths were defined based on the KEMAR dummy head  as shownInitially, the recorded signals were down-sampled to 22.05 kHz. The two microphones output signals were considered as:s is the clean speech, ix and iv  are the noise-corrupted speech and the wind noise for the ith microphone, respectively, and td is the time delay between the microphones.where j-3, j-2, j-1, and j, respectively. The RNN took acoustic features as its inputs and predicted the IRM  and iV represent the magnitudes of si(t) and vi(t) in the mth channel of frame j, respectively. Note that the clean speech recordings were used to obtain si(t). The microphone signals were segmented into frames with a duration of 5 ms (110 samples) and an overlap of 50% (55 samples) between successive frames. We used a 32-channel gammatone filter bank IRM, wind at 45°with speech at 90° , and wind at 315° with speech at 360° ; note that in the last condition, the speech came from in front, as 360° is equivalent to 0°. In all cases, the test data were recordings from BTE1. The total potential set of training data consisted of 320 seconds of male and female speech corrupted by 3 m/s wind noise, as received by the two BTE microphones for all eight pairs of wind and speech azimuths from  to  and for both BTE1 and BTE2 (16 20-second sets of two microphone recordings). The training data actually used were based on 13 of the 16 sets (excluding recordings from BTE1 for the three pairs of test azimuths), giving 260 seconds of training data.d′ . The root-mean-square input level (before frequency-dependent amplification for the hearing-impaired participants) of the speech (excluding the wind noise) was 60 dB SPL. Three conditions were used: noisy speech with no processing (NS), speech processed using the RNN, and high-pass filtered speech (HPF). The stimuli for the hearing-impaired participants were processed using linear frequency-dependent amplification according to the “Cambridge formula”  to ensurThe three conditions were compared in terms of subjective intelligibility and sound quality, using a paired-comparison procedure. There were three types of paired comparisons: NS versus RNN, RNN versus HPF, and NS versus HPF. The procedure was the same as described by The main experiment consisted of two parts. In the first part, participants were asked to indicate their preference in terms of subjective intelligibility. For this part, the instructions to the participant, which appeared on the computer screen, were as follows: “On each trial you will hear the same sentence twice in succession. Please decide whether the first or second sentence is more intelligible and by how much, by using the mouse to position the slider on the screen.”In the second part, participants indicated their preferences in terms of subjective sound quality and comfort. The instructions for this part were as follows: “On each trial you will hear the same sentence twice in succession. Please decide whether the first or second sentence is more comfortable and by how much, by using the mouse to position the slider on the screen.”On each trial, each pair of sounds was presented only once. Participants responded using a mouse to select the position of a slider on the screen along a continuum labeled “1 much better,” “1 moderately better,” “1 slightly better,” “equal,” “2 slightly better,” “2 moderately better,” and “2 much better.” Choices were not restricted to the labeled points; any point along the slider could be chosen. Within a given block of trials, each of the three pairs of conditions was presented twice in both orders for each of six sentences , so there were 72 trials in a block. There were six blocks per participant .Preference scores for each participant and each pair of conditions were computed in the following way. The extreme positions of the slider were arbitrarily assigned values of −3 and + 3. Regardless of the order of presentation in a given trial (Condition X first or Condition Y first), if X was preferred, the slider position was coded as a negative number and if Y was preferred, the slider position was coded as a positive number. For example, if the order on a given trial was Y first and X second, and the participant set the slider position midway between “2 slightly better” and “2 moderately better,” the score for that trial was assigned a value of –1.5. The overall score for a given comparison and a given azimuth was obtained by averaging the scores for the two orders for that comparison and azimuth for each participant. Scores were then averaged across participants, but separately for the normal-hearing and hearing-impaired participants. Therefore, preference scores were constrained to fall in the range −3 to + 3.d′  for all three test directions used, indicating high classification accuracy.To evaluate the accuracy of the RNN in estimating the IRM from the noisy speech signal, the estimated SNR for each time-frequency segment of the estimated IRM was compared with that for the IRM estimated using the clean speech signal. A classification threshold of 0 dB SNR was used to convert the IRM estimated by the RNN into a binary mask for the calculation of hit and false alarm rates . The hitd′  was calcd′ . The d′ W statistic was used, since the number of scores was small.To assess whether the preference scores were significantly different from zero (indicating a significant preference for one processing method relative to another), the scores for each participant were first averaged across azimuths. This was considered to be reasonable since the spectrum and level of the wind noise differed for each azimuth. Then, Wilcoxon nonparametric tests were used to assess whether the mean of the nine resulting scores for each pair of conditions was significantly different from zero (using two-tailed tests). The W = 15). For the comparison RNN versus NS (Panel b), the mean preference scores were all positive, favoring the RNN. The outcome of the Wilcoxon test was significant . For the comparison HPF versus NS (Panel c), the mean preference scores were positive for two azimuths and close to zero for the other. The outcome of the Wilcoxon test was not significant (W = 12). In summary, RNN was significantly preferred over NS, but HPF was not significantly preferred over NS. There was no significant difference between preferences for RNN and HPF.W = 6). For the comparison RNN versus NS (Panel b), the mean preference scores were all positive, favoring the RNN. The outcome of the Wilcoxon test was significant . For the comparison HPF versus NS (Panel c), two out of three preference scores were positive, favoring the HPF, but the score for azimuth 2 was very close to zero. The outcome of the Wilcoxon test was not significant (W = 6). In summary, RNN was significantly preferred over NS, but HPF was not significantly preferred over NS. There was no significant difference between preferences for RNN and HPF.W = 11). For the comparison RNN versus NS (Panel b), the mean preference scores were all positive, favoring the RNN. The outcome of the Wilcoxon test was significant . For the comparison HPF versus NS (Panel c), two out of three preference scores were positive, favoring the HPF, but the score for azimuth 2 was very close to zero. The outcome of the Wilcoxon test was not significant (W = 8). In summary, RNN was preferred over NS, but HPF was not significantly preferred over NS. There was no significant difference between preferences for RNN and HPF.W = 3, p < .05). For the comparison RNN versus NS (Panel b), the mean preference scores were all positive, favoring the RNN. The outcome of the Wilcoxon test was significant . For the comparison HPF versus NS (Panel c), two out of three preference scores were positive, favoring the HPF, but the score for azimuth 2 was very slightly negative. The outcome of the Wilcoxon test was not significant (W = 6). In summary, RNN was preferred over NS and HPF, but HPF was not significantly preferred over NS.p = .0002). Thus, for the results as a whole, there was a preference for RNN over HPF.The results showed small but significant preferences for RNN over NS for both subjective intelligibility and sound quality and for both groups. HPF was not significantly preferred over NS. Although there was only one significance difference between preferences for RNN and HPF in the individual Wilcoxon tests, the mean preference judgments for RNN versus HPF were positive for all azimuths for both groups for both subjective intelligibility and quality (12 cases), which is significant based on a binomial test , but was less accurate when the SNR was intermediate. Also some of the artifacts probably arose from the use of the original noisy phase in reconstructing the signal, which would make the speech sound noisy even if the SNR were estimated perfectly. Differences in preference across participants may reflect differences in the way that they weight the benefits of RNN noise reduction against the deleterious effects of the artifacts. The artifacts could potentially be reduced by setting a limit to the attenuation applied by the RNN or by limiting the speed of the gain changes, as is done in the noise-reduction systems of some commercial hearing aids . This reThe HPF processing did not result in the type of artifacts that occurred for the RNN, but it had the effect of removing the lower frequency components in the speech, making the speech sound “thin” or lacking in bass. It should be noted that the RNN used here operated “automatically.” Once trained, it was applied without any further adjustment of parameters. In principle, the RNN could be trained using recordings of wind noise obtained with a greater variety of wind speeds and azimuths and also with more different talkers than the two used here. Once trained, the RNN should automatically adjust its processing to deal with the changes in level and spectral content produced by differences in wind speed and azimuth, for example, produced by head movements. Furthermore, it should be possible to train the RNN so that it automatically does nothing when no wind noise is present. However, further work is needed to evaluate how well RNNs can be trained to generalize their performance to different wind speeds and talkers, including talkers not used for training.While the high-pass filtering used here was found to be marginally effective, the HPF cutoff frequency was chosen to be appropriate for the wind speed of 3 m/s actually used in the experiment. To apply HPF processing in a real hearing aid, it would be necessary to have an additional algorithm for detecting the frequencies at which wind noise was present and a method of selecting the appropriate high-pass cutoff frequency.The RNN processing was based on a relatively short frame duration of 5 ms, with a frame overlap of 50%. Ignoring limitations in signal-processing speed, the inherent delay produced by the RNN noise reduction would be about 7.5 ms, which is within the range that is acceptable for hearing aids , 2005. IOverall, the results suggest that processing using an RNN can be effective in reducing the effects of wind noise, and this has potential applications in hearing aids. Further research is needed to assess: (a) whether the benefits of RNN processing still occur when greater ranges of wind speeds and more talkers are used; (b) if better subjective intelligibility and quality are obtained when the amount and speed of wind noise reduction are limited to reduce artifacts; (c) if RNN processing is still beneficial when the RNN complexity is reduced to allow for the limited computational power of hearing aids.Two methods for reducing wind noise were evaluated. One method was a simple simulation of the processing that is often used in hearing aids and was based on HPF. The other method was based on the training of a RNN and the use of the trained network to reduce wind noise. The RNN was trained using recordings of the output of the two microphones of a BTE hearing aid in response to male and female speech at various azimuths in the presence of noise produced by wind from various azimuths with a velocity of 3 m/s, using the clean speech as a reference. A control condition using noisy unprocessed speech was also used (NS). A paired-comparison procedure was used to compare all possible combinations of the three conditions for subjective intelligibility and for sound quality or comfort. Eighteen native English-speaking participants were tested, nine with normal hearing and nine with mild-to-moderate hearing impairment. Frequency-dependent linear amplification was provided for the latter. Processing using the RNN was significantly preferred over no processing by both subject groups for both subjective intelligibility and sound quality, although the magnitude of the preferences was small. High-pass filtering was not significantly preferred over no processing. Although RNN was significantly preferred over HPF only for sound quality for the hearing-impaired participants, for the results as a whole, there was a preference for RNN over HPF. Overall, the results suggest that reduction of wind noise using an RNN is possible and might have beneficial effects when used in hearing aids.


A 43-year-old man presented to the emergency department with diabetic ketoacidosis, left facial pain, and swelling. Black eschar was noted to be protruding from both external nasal cavities. A computed tomographic (CT) scan was obtained, demonstrating sinus wall thickening and decreased attenuation of the left temporal lobe. Air fluid levels were also visualized in the sphenoid, left maxillary, and left ethmoid sinuses.What is mucormycosis?Are there specific associated risk factors?What are common presenting signs and symptoms?What are treatment options for rhino-orbital-cerebral mucormycosis?,Mucormycosis is a rare fungal infection, occurring mostly in immunocompromised patients, which can be potentially deadly. It is caused by the zygomycete, Mucorales, which are ubiquitous saprophytic fungi found in the soil and decomposing organic matter.1,3,4,6Immunocompromised patients are at the greatest risk for mucormycosis. Studies have shown that patients with hematologic malignancies, transplantation, neutropenia, corticosteroid therapy, and deferoxamine use are at an increased risk.1,5,7Bhansali et al4,Figs 2a and 2b). Regardless of patient complaints or findings on imaging, the gold standard for diagnosis remains biopsy and histopathology.For those patients unable to report signs/symptoms secondary to altered levels of consciousness, imaging can offer an alternative means for confirming clinical suspicion. CT findings include mucosal thickening and occupation of paranasal sinuses, with the ethmoid and maxillary sinuses being more frequently involved .2,7 CT s2-4,7,4,5,8Those patients who have a delayed diagnosis/treatment, hemiparesis/hemiplegia, bilateral sinus involvement, leukemia, or renal disease have a lower survival rate than their counterparts.1Our patient was found to have mucormycosis extending from his left nare all the way to the left foramen rotundum. After turbinate and maxillary sinus debridement by otolaryngology, orbital exenteration was required, given the extent of fungal spread. A preseptal plane was taken down to the orbital rims and then transitioned to a subperiosteal plane to ensure removal of all involved orbital soft tissues. The optic nerve was divided at the apex. The infraorbital nerve was sacrificed and debrided with its surrounding maxillary soft tissues. No maxillectomy was performed. The skull base and intracranial fungal extensions were treated via intrathecal and systemic amphotericin B.


Musical hallucination is a type of complex auditory hallucination. Possibleetiologies are deafness, psychiatric disorders such as schizophrenia, majordepression, use of medication and stress, besides neurologic diseases includingepilepsy, stroke and cancer. Uncommon etiologies encompass infectious diseases,metabolic disorders, and sensory deprivation. Although musical hallucinations have amajor impact on patients' lives, they have been undervalued and understudied in theliterature. We report a case of a 79-year-old woman with musical hallucination without cognitive impairment or hearing loss. Thepatient had preserved insight of her complaint and responded well toneuroleptics. Hallucinations are experiences similar to real perception yet with no external stimulus.They are vivid and bright, with all the strength and impact of normal perceptions, andare not under voluntary control. They can occur in any sensory modality, althoughauditory hallucinations are the most common in schizophrenia and related disorders.Musical hallucination (MH) is a complex auditory hallucination type described as hearingmusical tones, rhythms, harmonies and melodies without a corresponding external auditorystimuli in patients that are not necessarily affected by a psychopathological disorder.MH are often associated with serious hearing problems, although other causes exist. Somepatients never discover a definite cause. These hallucinations can be continuous orintermittent and occur in clear conscience and with preserved insight.MH are predominantly found in older women with progressive hearing loss secondary togeneral diseases or specific ear injuries. They also occur in neurological disorders,psychiatric disorders, especially depression and intoxication, as a sole cause or incombination. MH have been described following the use of tricyclic antidepressants, salicylates, benzodiazepines, pentoxifylline, propranolol,carbamazepine, phenytoin, alcohol abuse and general anesthesia.MH are underdiagnosed and undervalued by health professionals, despite the fact thatthese symptoms can significantly impair patients' quality of life. A small number ofstudies have been conducted to elucidate the pathophysiological process of MH, while fewstudies report successful treatment of these symptoms.MH were first described in the literature in the nineteenth century by Baillarger (1846)and Griessinger (1867).Incidence of MH is estimated as one new case per 10,000 persons per year withpsychiatric illness over the age of 65 years.The most accepted pathogenesis for MH associated with hearing loss and sensorydeprivation (caused by a cochlear injury or information interruption at the pons ormidbrain) is disinhibition of auditory memory circuits. Based on this prerogative, someauthors argue that MH can be a form of auditory Charles-Bonnet syndrome, causingabnormal activity in music processing cortical modular segments.We report the case of an elderly woman with preserved cognition and insight of herdisorder  and with no psychiatricdisorders.A 79-year-old woman with 11 years of formal education sought the geriatric outpatientfacility of the Faculty of Medicine of Jundiaí complaining of hearing the NationalAnthem sung 24 hours a day. During the daytime she took to turning the radio ortelevision up to a high volume to avoid hearing the anthem. At night the music becamemore intense making it difficult to sleep. After 3 months of persistence of thehallucination she became very anxious, stressed and irritated. She had preserved insightof her problem and reported no other types of hallucination, depressive symptoms, memoryor cognitive complaints. She had used bromazepam 3 mgpreviously for a sleep disorder andbegan using it again after this period. She slept for around 4 hours per night.Physical, psychiatric, and neurological examinations were normal. Her medical background consisted of systemic hypertension, dyslipidemia, sporadicvertigo diagnosed as labyrinthitis, and coronary disease. The patient underwent coronaryartery bypass surgery 2 years earlier. She also reported regular use of atorvastatin 20mg, metoprolol 50mg, enalapril with hydrochlorothiazide (20/12.5 mg), buffered aspirin100mg, and nicergoline 30mg. There was no family history of psychiatric illness.,Despite an absence of hearing complaints, the patient underwent an audiometry exam. Theaudiometry test was normal. Biochemical tests  and magnetic resonance of the brain werenormal. Complementary and neuropsychological evaluation consisted of the CambridgeExamination for Mental Disorders of the Elderly (CAMDEX) and a cognitive batterycomprising the Cambridge Cognitive Examination (CAMCOG),She was treated with risperidone 0.5 mg daily for 6 months and showed completeremission. At 6 months after withdrawal of the medication she exhibited no further MH.She has been followed thereafter with no relapses for 9 years.What calls attention in this case is that the patient sought a geriatric clinic with thechief complaint of hearing the National anthem sung, uninterrupted, 24 hours a day, formore than three months. We have conducted a systematic search on PubMed  and LILACS in abid to identify similar case reports. All 25 studies reported on PubMed involved elderlywith musical hallucinations associated with adverse reactions to medications, dementiaor cognitive decline, deafness or hearing impairment, and cerebrovascular disease. ,In most reported cases, MH is part of other psychiatric disorders as opposed to thechief complaint for which the patient seeks medical attention. Our patient repeatedlyasked her husband where the song was coming from, despite preserved insight. Griffithset al. reported that people with MH initially believed that music was actually playingsomewhere else .According to its origin, MH can be classified as functional or organic. The functionaltype is associated with psychopathological disorders with no apparent physical damage tothe brain or presence of hearing loss, such as schizophrenia, depression andobsessive-compulsive disorder.In the case reported, we did not observe the presence of affective symptoms or otherpsychotic manifestations. She presented with a high level of emotional stress andanxiety. She has a son with Down syndrome, age 50, whose care left her tired; herdaughter-in-law did not allow her to see the grandchildren owing to family rifts.Previously, at 45 years old, she had major depression and after remission presented nofurther episodes. After onset of MH, her quality of life had declined and she had becomemore distressed, but without clinical symptoms consistent with major depression. Shescored 6 on the Geriatric Depression Scale, which could classify her as having a milddepression, but did not score on the CAMDEX depression scale. Santos et al. reported aseries of 8 subjects from an ENT clinic with MH and variable degrees of depression.The patient showed no cognitive impairment during the 9-year follow-up or abnormality onbrain resonance imaging (MRI). She also did not present with hearing loss and heraudiometry was normal. Kasei et al. had also described a woman with normal audiometryand MRI who presented abrupt MH in the form of well-known songs.The pathogenesis and neuropsychological basis of MH is not fully established, but mightinvolve central auditory processing mechanisms. MH are mainly associated with hearingloss, psychiatric and neurological disorders  and intoxication, as a single cause or incombination. However, MH are also seen in healthy subjects. This phenomenon tends to beunderdiagnosed if not actively explored. In the literature, there are few reports ofhealthy patients with MH, especially without hearing or cognitive impairments.


Giardia infections afflict nearly 33% of people in developing countries, and 2% of the adult population in the developed world. This study describes the single cyclic nucleotide-specific phosphodiesterase (PDE) of G. lamblia and assesses PDE inhibitors as a new generation of anti-giardial drugs.Giardiasis is an intestinal infection correlated with poverty and poor drinking water quality, and treatment options are limited. According to the Center for Disease Control and Prevention, Giardia genome database identified a single gene coding for a class I PDE, GlPDE. The predicted protein sequence was analyzed in-silico to characterize its domain structure and catalytic domain. Enzymatic activity of GlPDE was established by complementation of a PDE-deficient Saccharomyces cerevisiae strain, and enzyme kinetics were characterized in soluble yeast lysates. The potency of known PDE inhibitors was tested against the activity of recombinant GlPDE expressed in yeast and against proliferating Giardia trophozoites. Finally, the localization of epitope-tagged and ectopically expressed GlPDE in Giardia cells was investigated.An extensive search of the Giardia encodes a class I PDE. Catalytically important residues are fully conserved between GlPDE and human PDEs, but sequence differences between their catalytic domains suggest that designing Giardia-specific inhibitors is feasible. Recombinant GlPDE hydrolyzes cAMP with a Km of 408 μM, and cGMP is not accepted as a substrate. A number of drugs exhibit a high degree of correlation between their potency against the recombinant enzyme and their inhibition of trophozoite proliferation in culture. Epitope-tagged GlPDE localizes as dots in a pattern reminiscent of mitosomes and to the perinuclear region in Giardia.G. lamblia PDE activity leads to a profound inhibition of parasite proliferation and that GlPDE is a promising target for developing novel anti-giardial drugs.Our data strongly suggest that inhibition of  Giardia lamblia contains only one single PDE, named GlPDE. GlPDE activity is highly impaired by a range of PDE inhibitors, which also suppress parasite proliferation in vitro. Thus, there is a good agreement between PDE inhibition and parasite drug susceptibility. We demonstrate molecular differences between human PDEs and GlPDE that can be exploited for the development of GlPDE-selective inhibitors. Finally, our data may suggest localization of GlPDE to mitosome organelles, which are absent in human cells and thus are in the focus as possible targets for the treatment of giardiasis. This may add to the notion that GlPDE represents a potential target for the development of novel anti-giardial drugs.Cellular signaling by the cyclic nucleotides cAMP and cGMP is ubiquitously found in organisms from human to unicellular parasites. Cyclic nucleotide-specific phosphodiesterases (PDEs) are pivotal regulators of these signaling processes and these enzymes represent important drug targets for a variety of diseases. Eleven PDE families are distinguished in humans and selective inhibition of a single human PDE family without targeting others is feasible. In parasites, interference in the signaling mechanism by PDE inhibition may be fatal. The diarrhea-causing parasite  Giardia lamblia is a protozoan parasite that causes giardiasis, an intestinal disease with symptoms such as diarrhea, nausea, and malabsorption -x-H-x-[AG]-x(2)-[NQ]-x-[LIVMFY] , H-x-H-L-D-H-[LIVM]-x-[GS]-[LIVMA]-[LIVM](2)-x-S-[AP] , D-x-G-D-x-G-N-H-[ED]-x-H-x-G-H-x-H .Sequence patterns searches were done with the programs fuzzpro and patmatdb  were created using MSAprobs   trophozoites were grown under microaerophilic conditions in 11 ml culture tubes  containing TYI-S- 33 medium supplemented with 10% bovine serum and bovine bile according to standard protocols [rotocols . Parasitrotocols  by cultiG. lamblia WBC6 and the primer pair GlPDE1cat-for (5'-GCAGTCGACATATGGGAGTGGATTGTTCAAATATCAAGTTGG-3') and GlPDE1cat-rev (5'-CGTGGATCCTATTACTCCTTCTTGTCCAGCTCAATCTC-3'). The PCR product was then cloned into the TA vector pCRII-TOPO (Invitrogen) yielding plasmid pCR-GlPDE1cat. To get a plasmid containing the sequence coding for the full length protein lacking the N-terminal transmembrane helices (Met-588 –Glu-1371) of GlPDE, a further PCR product was produced using the primer pair GlPDE1-Mfor (5'-GAGCAGTCGACATATGCCGTGTGTTTTGTTTCGACTGG-3') and GlPDE1-LMrev (5'-GGTCTGCATGAGAACAGTACACGGTACTGAGCATG-3') and was cloned via NdeI (bold) and BsrGI into pCR-GlPDE1cat resulting in plasmid pCR-GlPDE1M. The inserts of both plasmid constructs were verified by sequencing and subcloned into two variants of the yeast expression vector pLT1 [Giardia protein alone, whereas the other adds an N-terminal hemagglutinin (HA) tag to allow detection of the recombinant protein. Transformation of the constructs into the PDE-deficient S. cerevisiae strain PP5 (MATa leu2-3 leu2- 112 ura3-52 his3-532 his4 cam pde1::URA3 pde2::HIS3) was carried out as described previously [The DNA sequence comprising the catalytic domain (residues Gly-983 –Glu-1371) was amplified by PCR using genomic DNA of tor pLT1  using theviously .S. cerevisiae strain PP5 was carried out exactly as described [The heat-shock assay to detect complementation of the PDE-deficient phenotype of the escribed . Yeast sYeast cell pellets were created exactly as described previously . BrieflyPDE activity was determined by a modification of the two-step procedure of Thompson and Appleman  as descrGiardia culture medium. G. lamblia WBC6 trophozoites were grown until adherent cells reached a confluency close to 100%. Cells were then chilled on ice for 20 min and adherent parasites were detached from the culture tube. Cultures were diluted to 5x103 cells/ml in growth medium (room temperature) and 100 μl aliquots were immediately added to wells of 96-well plates preloaded with the same volume of test compound dilutions. DMSO (solvent) controls were always included. Plates were incubated for 72 h at 37°C in a humid chamber with an anaerobic atmosphere. Adherent cells were then washed twice with 200 μl PBS before adding 200 μl of PBS containing 10 mg/ml resazurin and 1% glucose. After incubation for 4 h at 37°C, plates were read on a fluorescence plate reader  with excitation and emission wavelengths of 544 nm and 590 nm, respectively. Data were analyzed with the GraphPad Prism software package using a sigmoidal dose-response model for regression. All assays were done in quadruplicate and at least three independent experiments were performed.Test compounds were dissolved in DMSO and serially diluted in G. lamblia trophozoites as follows: The entire open reading frame of GlPDE (gene ID GL50803_14058) and 43bp of the 5’-upstream region were PCR amplified from genomic DNA using the primer pair 5’-CGTCTAGATTTGTGTCATAAGCAAGGTAA-3’ and 5’-CGTTAATTAACTACGCGTAGTCTGGGACATCGTATGGGTACTCCTTCTTGTCCAGCTCAA-3’. These primers introduce a C-terminal HA-tag (underlined) and the two restrictions sites XbaI (bold) and PacI  into the amplicon. The XbaI/PacI-digested PCR product was cloned into vector PAC-CHA [Giardia WBC6 trophozoites were transfected by electroporation using 15 μg of plasmid DNA as input . Stable transfectants were selected with the antibiotic puromycin  at a concentration of 77 μM. Expression of the PDE is driven by its endogenous promoter in the upstream region. The transiently transfected cells, expressing HA-tagged GlPDE under its endogenous promoter, were used in all localization studies.C-terminally HA-tagged GlPDE was expressed under its endogenous promotor in  PAC-CHA  and GiarEndocytic uptake of the fluorescent dye “dextran Alexa Fluor 594”  by PV organelles was achieved as described previously . BrieflyRNA was isolated from wild type cells using an RNAeasy kit (QIAGEN Cat. 74104) following the ‘Animal Cells Spin’ protocol. A DNase I digestion was included to remove residual genomic DNA according to the manufacturer’s protocol. RNA was eluted in 50 μl of RNase-free water. Reverse transcription was performed using a Qiagen Omniscript RT kit (Cat. 205111) and cDNA was amplified by PCR using different combination of primers.Giardia Genome Database (www.GiardiaDB.org version 4.0) [Giardia is a highly diverged organism [G. lamblia isolates sequenced so far . A putative PDE with similarity to GlPDE was also identified in the database of annotated proteins of the salmon parasite Spironucleus salmonicida, a diplomonad species such as G. lamblia. GlPDE and the Spironucleus PDE  share a similar domain structure, a short stretch of weak homology in the N-terminal protein half (185 aa with 24% identity) and 35% identity in the C-terminal catalytic domain, indicating that both enzymes are distantly related members of the same PDE family   were annorganism . To enhamily see . To idenHMMs see , but no eq study . In contGiardia genome assemblages A, B and E [Giardia genotypes, with the only exception of Ser-1316 that is replaced by a leucine in isolate P15 of the non-human infective assemblage E (The open reading frame of GlPDE GL50803_14058) encodes a protein of 1371 amino acids  at this position . In variGiardia assemblages A, B and E . These two residues substitute a totally conserved acidic residue  and an invariant histidine of human PDEs, respectively , which h B and E .S. cerevisiae strains lacking both endogenous PDEs display heat-shock sensitivity as a phenotype [henotype . Functiohenotype ,43,42. Shenotype . As predm of 408 ± 37 μM (Soluble lysates prepared from the recombinant yeasts expressing GlPDE(aa588-1371) hydrolyzed cAMP with standard Michaelis-Menten kinetics with a K ± 37 μM . The enzG. lamblia trophozoites in culture . This profiling assay showed that most of the hPDE-family-specific inhibitors were essentially inactive against recombinant GlPDE, even at 100 μM concentration . In cont culture . At 50 μactivity . RepreseG.lamblia trophozoites, parasites were transfected with a plasmid encoding a C-terminally HA-tagged copy of GlPDE plus the 200 bp upstream region containing the putative endogenous promotor, resulting in the transiently transfected strain PDEp-PDEHA. The subcellular localization of the chimeric protein was detected by immunofluorescence staining in formaldehyde-fixed cells using an anti-HA antibody. The HA-tagged GlPDE was predominantly detected in dot-like patterns in the trophozoite periphery, and at distinct dots that are centrally located between the two nuclei. This localization pattern may indicate that GlPDE is associated with the various peripheral and the centrally located mitosomes. Further staining was found in the region surrounding the Giardia nuclei, which is reminiscent for the endoplasmic reticulum , followed by fixation and staining with anti-HA antibodies and a secondary antibody conjugated to Alexa Fluor 488 (green) , the transiently transfected een) see . MicroscG. lamblia, GlPDE, and it addresses the question whether this enzyme could represent a valid drug target. The sequence characteristics unambiguously classify GlPDE as a new member of the class I PDE superfamily that includes all human and many protozoan parasite PDEs [The present study reports on the identification and characterization of the single PDE from ite PDEs ,27. GlPDThe N-terminal half of the protein is predicted to contain eight or nine putative transmembrane helices (TMH), of which the first two are predicted to represent a mitochondrial targeting signal. The TMH region is followed by a stretch of about 400 amino acids with no discernible domains, and a C-terminal catalytic domain with a high degree of similarity to those of the human PDEs.Plasmodium falciparum PDEs [The presence of such an extended N-terminal transmembrane domain containing two long extra-membranous regions (~140aa) is a feature that has not been described in other PDEs to date, though membrane-anchoring through a TMH bundle is also found in many other PDEs such as in hPDE3  and in arum PDEs .Giardia trophozoites suggests a localization at mitosomes. Mitosomes are organelles that are found in unicellular microaerophilic or anaerobic organisms such as Entamoeba, microsporidia and Giardia. They are believed to be derived from mitochondria, but lack the capability to gain energy from oxidative phosphorylation [Giardia and presumably regulated by cAMP signaling, future studies to verify the localization of GlPDE are certainly warranted.The prediction of an N-terminal mitochondrial targeting signal in GlPDE is in agreement with our finding, that HA-tagged GlPDE transiently expressed in rylation ,72. A nurylation . Our tenrylation ,75. Sincm for cAMP of 408 μM is much higher when compared with hPDEs, where values do not exceed 20 μM for the preferred cyclic nucleotide [m for their preferred substrate have been identified in other lower eukaryotes: For the PDEA orthologs of various kinetoplastid parasites, Km values between 190 and 600 μM have been reported [With respect to the catalytic domain, all residues important for substrate recognition and catalysis are conserved between GlPDE and the consensus of human PDEs . Substracleotide . Althougreported ,43,79.m of recombinant GlPDE(aa588-1371) for cAMP indicates that cAMP hydrolysis in Giardia occurs with first-order kinetics over a wide concentration range. The presence of a high-Km PDE as the sole enzyme for cyclic nucleotide degradation is somewhat unusual. However, a similar case is observed for the fission yeast Schizosaccharomyces pombe. Its genome codes only for a single class II PDE, Cgs2, that exhibits a high Km for cAMP. Nevertheless, this single PDE apparently is sufficient for all aspects of cAMP management, a situation akin to G. lamblia [The high K lamblia ,81.m PDEs usually also express a low-Km enzyme, though their distinct roles are often not clearly understood. In the yeast S. cerevisiae, the high-Km, class II PDE, pde1, is the major regulator of agonist-induced cAMP signaling, while the low-Km, class I PDE, pde2, has only a limited effect on this pathway [Dictyostelium discoideum, several discrete pools of cAMP exist in the cell, and each is addressed by a specific pair of PDEs, one of high Km and low capacity, and the second of low Km and high capacity. In combination, they are thought to keep the average half-life of cAMP down, irrespective of fluctuating local concentrations [Magnaporthe oryzae, the high-Km PdeH and the low-Km PdeL play unique and non-redundant roles, with PdeH involved in conidial morphogenesis and PdeL in pathogenic development [Other unicellular eukaryotes that express high-K pathway . In the trations . In the elopment ,85.Giardia trophozoite proliferation. Interestingly, the compounds with good correlations of biochemical and cell-biological activities represent four different chemical scaffolds, and thus represent an excellent starting base for their further development into Giardia-specific PDE inhibitors and potential drug candidates. In addition, the amino acid sequence differences, and—by inference—the corresponding structural differences, between human and Giardia PDEs, indicate that a discrimination between human PDEs and GIPDE by appropriate structure optimization of drug candidates is feasible.In order to pharmacologically evaluate the role of GlPDE, a panel of established PDE inhibitors with activity against human and/or trypanosomal PDEs was screened against recombinant GlPDE . We identified a number of compounds that inhibited GlPDE in the low micromolar or sub-micromolar concentration range. All these inhibitors, with the exception of dipyridamole, also impaired Giardia reduce nitro-groups to toxic nitroso derivatives, as it is the case for metronidazole, the current first-line drug against giardiasis [Not surprisingly, a few compounds of the original set exhibited an effect on trophozoite proliferation, but did not inhibit GlPDE enzyme activity, indicating off-target effects. It could also mean that such compound targets only the native full-length PDE in the parasite and not the heterologously expressed GlPDE(aa588-1371) used in the screenings. However, this is rather unlikely, since almost all known PDE inhibitors target the PDE catalytic pocket . In the ardiasis . Off-tarardiasis –89. Thusardiasis .G. lamblia expresses only a single PDE, which we here characterized on the molecular and functional level. A first pharmacological validation has shown that targeting GlPDE activity leads to significant impairment of G. lamblia throphozoite proliferation in vitro, which would be an important prerequisite to alleviate giardial disease symptoms. These promising findings warrant further studies in vitro and in vivo to exploit GlPDE as a potential target for the treatment of giardiasis.In conclusion, we show that http://www.giardiadb.org) accession numbers for the gene products discussed in this paper are as follows: (i) GlPDE homologs in different Giardia isolates (assemblage/isolate): A/WB (GL50803_14058), A/DH (DHA2_150867), B/GS (GL50581_303), B/GS_B (GSB_153349), E/P15 (GLP15_4333); (ii) identified putative nucleotidyl cyclases (classIII) in assemblage A, isolate WB: strong hit (GL50803_14367), strong hit (GL50803_16599), weak hit (GL50803_16492); (ii) protein kinase A subunits : catalytic subunit gPKAc (GL50803_11214), regulatory subunit gPKAr (GL50803_9117); (iii) putative phosphodiesterase SsPDE of S. salmonicida strain ATCC50377 (SS50377_13952).The GiardiaDB Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.S5 Fig(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S1 File(PDF)Click here for additional data file.


F = 7.516, P = 0.000). Furthermore, we found that the prognosis of tinnitus was not related to the type of audiogram or level of hearing loss. Recovery from a severe level of hearing loss was, however, found to be associated with a poor tinnitus prognosis . In summary, our study indicates that the association between tinnitus and SSNHL is extremely high. Tinnitus can be ameliorated by the successful treatment of hearing loss. The study was registered in the Chinese Clinical Trial Registry (ChiCTR1800014797).Tinnitus is believed to result from the maladaptive plasticity of the auditory nervous system; reports regarding its severity and prognosis are conflicting. We evaluated the characteristic and short-term prognosis of tinnitus associated with sudden sensorineural hearing loss (SSNHL). A total of 230 cases were enrolled. The severity and 1-month prognosis of tinnitus (according to the Tinnitus Handicap Inventory (THI)) were assessed in terms of the patients' sex, age, level of hearing loss, type of audiogram results, and so on. According to our statistical analysis, the degree of handicap due to tinnitus was not related to sex, age, or level of hearing loss; the Tinnitus Handicap Inventory indicated that the low-frequency-audiogram group had a low tinnitus handicap ( Tinnitus, one of the most frequent sensorineural disorders, involves the perception of a fake sound in the absence of a corresponding sound stimulus; it is often considered as the result of maladaptive plasticity in the auditory system , 2. It iThe purpose of the present research was to estimate the characteristics and prognosis of tinnitus associated with SSNHL and discuss the role of neural plasticity in tinnitus.This research was approved by the Zhongda Hospital Southeast University Research Ethics Committee . Fully informed written consent for publication of clinical data was taken from each patient or from the guardians of the patients who were below 18 years of age.A retrospective study was applied in patients suffering from tinnitus triggered by unilateral SSNHL who were treated at our hospital between January 2015 and July 2017; those with newly developed tinnitus were enrolled in our study. SSNHL was defined as more than 30 dB HL threshold shift in three contiguous frequencies or more in 72 hours. The cause of hearing loss was unknown; enrolled patients suffering from unilateral SSNHL were treated within 14 days of onset. The hearing loss and tinnitus were assessed before treatment and in the following 30 days.Ginkgo biloba leaves injection, Dr. Willmar Schwabe Pharmaceuticals) administration. They were treated with hyperbaric oxygen therapy for 60 minutes once daily for 10 days, intravenous hydrocortisone sodium succinate , ginaton (87.5 mg/day for 10 days), and lidocaine (100 mg/day for 10 days). Patients with diabetes or hypertension were treated by intratympanic injection of prednol (20 mg/day for 10 days) instead of intravenous hydrocortisone.All patients received hyperbaric oxygen therapy, steroid, lidocaine, and ginaton  being one of the validated questionnaires. The THI questionnaire consists of three subscales: emotional 9 items), functional 11 items), and catastrophic (5 items). The yes answer to an item gets 4 scores or sometimes 2 scores, and a no gets a zero score. Scores of the total scale range from 0 to 100, with higher scores representing a greater perceived handicap [ items, f items, aPure-tone threshold was measured in the affected and nonaffected ears. Pure-tone average thresholds at 0.5, 1, 2, and 4 kHz were assessed as mean pure-tone threshold. When the hearing thresholds of deep losses were not detected, the threshold was considered as the maximum audiometric intensity. Hearing loss was classified into 5 degrees according to the mean pure-tone threshold: mild (26–40 dB HL), moderate (41–55 dB HL), moderately severe (56–70 dB HL), severe (71–90 dB HL), and profound (>90 dB HL). Pure-tone threshold was measured before treatment and during the 30-day period after onset.The audiogram was classified into 4 types by the method reported in. The audThe following criteria  were useStatistical analyses were performed with SPSS software . Tests and graphs were based on analysis sets that included all patients. The severity of tinnitus (THI) in terms of patients' sex, age, audiogram type, and hearing loss level was studied by Ridit analysis. The prognosis of tinnitus in terms of audiogram type, level of hearing loss, prognosis of hearing loss, and the severity of tinnitus (THI) was also studied by the same method.Of a total of 283 unilateral SSNHL patients enrolled within 14 days of the onset of acute hearing loss, 252 (89.0%) reported the new development of tinnitus. Of these 252, a total of 22 patients were lost to follow-up. The mean age of the 230 assessed patients was 43.5 years, with a median of 45 years, standard deviation of 15.1; minimum of 15 years and maximum of 70 years. Of those assessed, 136 patients were men (59.1%) and 94 were women (40.9%). There were 22 cases of negligible tinnitus (9.57%), 96 mild cases (41.74%), 86 moderate cases (37.39%), 22 severe cases (9.57%), and 4 catastrophic cases (1.74%).P = 0.600). There was also no significant difference between age groups (P = 0.598).Sex, age, initial hearing loss level, and audiogram type were reported to be associated with the severity of tinnitus. The degrees of difference in THI in terms of sex and age were compared by Ridit analysis . The patF = 0.704, P = 0.550). However, there was a significant difference among different audiograms . The low-frequency-audiogram group had a lower score on the THI compared with others.The THI and audiogram scores at different levels of hearing loss were compared by Ridit analysis . In termF = 1.640, P = 0.181) . Patients of the no effective group had a poor tinnitus prognosis compared to the cure group and effective group  and 48 noneffective cases (20.87%). The tinnitus prognosis was compared in terms of audiogram result, hearing loss level, hearing prognosis, and THI score. There was no significant difference in tinnitus prognosis among the different audiogram types (= 0.181) . Howeverve group .F = 0.170, P = 0.917) . The initial degree of THI was also compared with the final degree of THI. Patients with moderate and severe degrees of THI had better tinnitus prognosis than others  . Howevern others .The generation and maintenance of tinnitus are challenging topics of neural research , 13. TheIn most patients, tinnitus is associated with hearing loss , 15, 16.The severity of tinnitus can be influenced by cochlea damage-induced neuronal plasticity throughout the auditory nervous system from the hair cells to the auditory cortex  and alsoIt has been found that tinnitus can be treated effectively by treating the concomitant conductive hearing loss –31. TinnOur study found that hearing recovery is consistent with a decrease in tinnitus, although the initial level of hearing loss or audiogram type is not a prognostic factor for tinnitus. A study has also shown earlier effects on hearing recovery than on complete tinnitus remission in patients with severe-profound hearing loss . HearingThe results of the present study suggest that the association between tinnitus and SSNHL is extremely high. Tinnitus associated with SSNHL has a negative impact on patients' quality of life. The tinnitus was less severe in the low-frequency-audiogram group compared with other types. Tinnitus can be ameliorated by the treatment of SSNHL. Maladaptive plasticity plays an important role in the mechanism of tinnitus. However, the present research reports the short-term prognosis of tinnitus as associated with SSNHL; the longer-term prognosis requires further analysis to assess the neural plasticity of the cochlea and the brain.


Aspergillus fumigatus. Following purification, this enzyme exhibited a specific activity of 1036.76 U/mg protein, a molecular weight of 9.5 kDa, and an optimal temperature and pH for enzyme activity of 60°C and 5.0, respectively. However, its activity was partially inhibited by Fe3+, Cu2+, and Zn2+, but slightly improved by the presence of K+ and Na+. Additionally, chemical-modification experiments were also applied to investigate the structural information of 5'-PDE, in which the residues containing carboxyl and imidazole groups were essential for enzyme activity based on their localization in the 5′-PDE active site. Furthermore, purified 5′-PDE could specifically catalyze the synthesis of ribonucleotides with a Vmax 0.71 mmol/mg·min and a KM of 13.60 mg/mL.5′-Phosphodiesterase (5′-PDE) catalyzes the hydrolysis of ribonucleic acid to obtain a mixture of ribonucleotides, such as 5′-guanosine monophosphate and 5′-adenosine monophosphate. In this study, a 5'-PDE was newly isolated and purified from  Recently, interest in 5′ nucleotides has increased due to their applications in food and the pharmaceutical industries . As a flPenicillium citrinum  was used to detect the effect of substrate on EDC regarding 5′-PDE activity.Different concentrations of TNBS  were dissolved in 100 mM phosphate buffer (pH 8.0), and the enzyme solution (0.1 mL) was incubated with substrate solution (0.1 mL TNBS and 0.8 mL phosphate buffer) at 30°C for 30 min. The remaining activity of 5′-PDE at each point was normalized as the percentage of enzyme activity in the absence of TNBS. Similarly, the reaction system  was applied to detect the effect of substrate on TNBS regarding 5'-PDE.N-ethylmaleimide , phenylmethanesulfonyl fluoride , and 2-hydroxy-1-ethanethiol  were also applied to modify 5′-PDE activity to investigate the structural characteristics of 5′-PDE.Similarly, different concentrations of diethyl oxydiformate , 2,3-butanedione , 2PO4 used as the mobile phase at a flow rate of 0.6 mL/min. All experiments were performed in triplicate, and data are represented as the average of three replicates, with errors indicating the standard error of the mean.The total concentrations of AMP, GMP, and UMP in the reaction mixture were determined by high-performance liquid chromatography  equipped with a Synergi Fusion-RP C18 column (250 × 4.60 mm) and an ultraviolet detector (λ = 260 nm), with 0.1 mol/L KHA. fumigatus SCSGAF0014 (JN850983) and A. fumigatus (HQ149773)  . PhylogeTo optimize 5′-PDE production, different factors, including carbon source, nitrogen source and temperature, were investigated during preliminary experiments, where glucose and water content affected 5′-PDE production. Therefore, different levels of glucose and water content were employed, resulting in maximum 5′-PDE titers from 40 g/L glucose and 45% water content of 3753.4 U/g and 4512.6 U/g, respectivity . SubsequTo obtain the higher degree of 5′-PDE purity, 5′-PDE purification was performed by microfiltration, ultrafiltration, ammonium sulfate precipitation, DEAE-cellulose column chromatography, and DEAE-sepharose CL-6B chromatography . FollowiAs shown in 2+, Fe3+, and Cu2+ inhibited 5′-PDE activity, whereas K+ and Na+ slightly improved 5′-PDE activity. We observed that 0.1 mol/L Fe2+ or Cu2+ significantly decreased 5′-PDE activity by 34.5% and 26.2%, respectively. Under optimal conditions , 5′-PDE kinetic parameters were measured using from 1.0 mg/mL to 12.0 mg/mL of RNA as substrate, resulting in apparent KM and Vmax values of 13.60 mg/mL and 0.71 mmol/mg·min, respectively  suggested that one carboxyl group located in the 5′-PDE active site .Similarly, DEPC was also used to modify imidazole groups present in histidine residues under neutral conditions. Our findings demonstrated that one imidazole group was present the active site and essential for 5′-PDE activity .As shown in To evaluate 5′-PDE catalytic characteristic, hydrolysis experiments were performed by HPLC using RNA as the substrate. As shown in A. fumigatus, followed by analysis of important structural characteristics necessary for RNA hydrolysis. Following purification, 5′-PDE exhibited a specific activity of 1036.76 U/mg protein and a molecular weight of 9.5 kDa, as well as optimal temperature and pH of 60°C and 5.0, respectively. However, 5′-PDE activity was partially inhibited by the presence of Fe3+, Cu2+, Fe2+, and Zn2+, but slightly improved by K+ and Na+, which agreed with a previous report [Vmax and KM values of 0.71 mmol/mg·min and 13.60 mg/mL, respectively, which were superior to those of nuclease P1 from P. citrinum (KM was 35.44 mg/mL) [A. fumigatus, including: (1) the low efficiency of free-cell fermentation for 5'-PDE production; (2) the security problems for 5′-PDE from A. fumigatus[In this study, we described the isolation of purification of 5′-PDE with excellent catalytic specificity from s report . Based o4 mg/mL)  and indifumigatus.P. citrinum were effectively improved, which subsequently reduced the cost of nuclease P1 production [Therefore, to improve fermentation performance, immobilized fermentation were proposed and applied to produce 5′-PDE . Compareoduction . HoweverP. citrinum, an anamorphic mesophilic fungus with a history of safe use as a fermentation organism in Europe and Asia, is used in the production of ribonucleases [A. fumigatus, a pathogenic bacteria, for use in food processing should be comprehensively evaluated by means of standard toxicological testing methods, including in vitro Ames tests and chromosome-aberration assays and in vivo rat erythrocyte micronucleus assays, in future studies.Currently, 5′-PDE has potential commercial applications in the food industry, specifically in the processing of nucleotide-rich food ingredients to enhance flavors in food products, such as umami. Therefore, security problem played an important role in industrial applications for 5′-PDE . Nucleasucleases ; howeverS1 Fig(a) the amplification of 18S rDNA; (b) the phylogenetic analysis of strain XD-9.(DOCX)Click here for additional data file.S2 FigEffects of glucose (a) and water content (b) on the enzyme activity of 5'-PDE.(DOCX)Click here for additional data file.S3 Fig(DOCX)Click here for additional data file.