To compare the development of the auditory system in hearing and completely acoustically deprived animals, naive congenitally deaf white cats (CDCs) and hearing controls (HCs) were investigated at different developmental stages from birth till adulthood. The CDCs had no hearing experience before the acute experiment. In both groups of animals, responses to cochlear implant stimulation were acutely assessed. Electrically evoked auditory brainstem responses (E-ABRs) were recorded with monopolar stimulation at different current levels. CDCs demonstrated extensive development of E-ABRs, from first signs of responses at postnatal (p.n.) day 3 through appearance of all waves of brainstem response at day 8 p.n. to mature responses around day 90 p.n.. Wave I of E-ABRs could not be distinguished from the artifact in majority of CDCs, whereas in HCs, it was clearly separated from the stimulus artifact. Waves II, III, and IV demonstrated higher thresholds in CDCs, whereas this difference was not found for wave V. Amplitudes of wave III were significantly higher in HCs, whereas wave V amplitudes were significantly higher in CDCs. No differences in latencies were observed between the animal groups. These data demonstrate significant postnatal subcortical development in absence of hearing, and also divergent effects of deafness on early waves II–IV and wave V of the E-ABR. The auditory system demonstrates extensive developmental changes during postnatal life, both in humans as well as in altricial animals (review in ). Which An interesting model in resolving this question is the congenitally deaf cat . CDCs sExtensive changes in the auditory cortex have been demonstrated in adult CDCs  as well The present study was designed to investigate the consequences of inborn deafness on the brainstem function. In auditorily naive animals of different ages, brainstem-evoked signals were measured in response to electrical stimulation through a cochlear implant. The responses were compared to electrically evoked brainstem responses in hearing (acutely deafened) controls. Electrically-evoked auditory brainstem responses (E-ABRs) are also used clinically in human cochlear implanted subjects to objectively assess the auditory function –20. The The present data reveal that auditory deprivation does affect the brainstem and that these functional effects can be detected using E-ABRs. The data demonstrate that a significant portion of the developmental process is, however, preserved in complete deafness. Consequently, this part of development is dependent on genetic makeup. Finally, the present data show that different portions of the afferent auditory pathway are differentially sensitive to deprivation.For the present study, 35 cats were investigated. Their age was in the range from birth (P0) to adult (>6 months), 17 animals were congenitally deaf and 18 were normal hearing controls. All congenitally deaf cats had no hearing experience before the final (acute) experiment. Two animals from the deaf white cat colony were investigated at 0 p.n. (right after birth), one deaf animal at day 3 p.n. (P3) and one deaf animal at P8, remaining animals were older than 21 days. Congenital deafness was, however, verified in the 4th week p.n. by the absence of acoustically evoked brainstem responses (see .μs) were used. Stimulation was performed at intensities in the range of 5–120 dB SPL, presented at a rate of 13 stimuli per second. For recording of auditory brainstem responses (ABRs), a small trephination was drilled at the vertex of the skull and a recording electrode was placed epidurally. The indifferent electrode was inserted in the muscles under the bulla at the site of stimulation. The recorded signal was amplified by 80 dB (Tektronix V122 and 5A22N), filtered , and averaged by a computer (100 repetitions). Animals classified as hearing had a click-evoked ABR threshold <40 dB SPL.All animals were anaesthetized by subcutaneous application of ketamine-hydrochloride  and xylazine-hydrochloride . The pinna was removed at both sides, the tympanic membranes and the bullea were exposed. The animals were placed in a soundproof chamber and fixated in a stereotactic frame. For acoustic stimulation, an inversely driven calibrated Bruel and Kjear condenser microphone was placed in front of the tympanic membrane. For assessing hearing thresholds, condensation clicks  was performed using an optically isolated current source. Presentation rate was 13 stimuli per second in all animals. Electrically evoked brainstem responses were recorded at varying intensities. Stimulus levels were computed from peak-to-peak amplitudes of the pulses. The highest current level was reached when facial nerve stimulation appeared. Recordings with facial nerve stimulation resulting in muscle contractions were contaminated with muscle activity and discarded from further processing.After determining the hearing thresholds, both bullae and round windows were opened. In hearing controls the hair cells were destroyed using a slow instillation (within 5 minutes) of 0.3 mL neomycine sulfate (25 mg/mL) into the scala tympani. This procedure was performed to avoid electrical stimulation of hair cells (electrophonic effects). After further 5 minutes, the neomycine was carefully washed out by Ringer's solution and deafness was verified by the absence of auditory-evoked brainstem responses . The deafening procedure was performed on both ears. Thereafter, a custom-made cochlear implant with five intrascalar gold contacts with a spacing of 1 mm  was placI-V based on morphology and latency  were designated  latency . Threshoα = 10%), similarity of the distribution using the f-test (α = 10%) and final comparisons using t-tests (two-tailed tests α = 5%).Normality was tested using the Pearson-Stephens test  was confirmed in the acute experiment.Electrically evoked brainstem evoked responses (E-ABRs) in adult animals revealed a morphology characteristic of acoustically evoked brainstem response . In mostNone of these four animals revealed any ABRs up to click levels of 120 dB SPL, even though the ear canals have been surgically removed, the tympanic membrane was exposed and the stimulation was performed using a closed system.Individual fissures of the skull were not yet ossified in this group of animals. The external meati were closed. At P3 and P8, however, they already started to open from the round widow side. Consequently, at P3 and P8, close to the tympanic membrane, the walls of the meatus did not adhere to each other anymore and the closed portion was few millimeters distant from the tympanic membrane. The middle ear was not pneumatized in this age group. The bulla was filled with a viscous whitish tissue that did not adhere to the bulla walls nor to the round window . It coulStimulation with a cochlear implant allowed a controlled and comparable testing of animals despite a nonfunctional cochlea. Using monopolar configuration, the maximal portion of the auditory nerve was stimulated and positional effects were minimized.Substantial maturation of E-ABRs was observed within the first days of life . At P0, The range of suprathreshold intensities that were possible to test differed in individual animals due to muscular artifacts. These were generated by facial nerve stimulation at high current levels. Consequently, the dynamic range of E-ABRs that was possible to evaluate varied between 4 and >10 dB above threshold. To include all investigated animals, individual E-ABRs waves were compared at 4 dB above thresholds. Only positive peaks of the E-ABR components were processed.P = 0.0489; wave II  P = 0.00115; wave III  P = 0.030; wave IV  P = 0.012; wave V  P = 0.021; two-tailed t-test), demonstrating a developmental change. In deaf animals, the developmental change was similar, whereas for wave II, the differences did not reach the level of statistical significance . In HCs and CDCs, the timecourse of development of brainstem evoked response latencies was similar . Already at threshold intensity, E-ABRs showed most frequently waves III/IV and V. However, in some animals, also (sometime only) earlier waves  or later waves  appeared at threshold. To compare the E-ABRs in a wave-specific manner, the thresholds were also compared on the basis of “wave thresholds” . This result indicates that potentially divergent deprivation effects take place in the generators of the early waves II–IV and the wave V. Additionally, deaf cats consistently demonstrated higher interindividual variance in thresholds.Lowest E-ABR thresholds did not significantly differ between hearing and deaf animals if all waves were included  and wave V . Also in this respect, a discrepant effect of deprivation was observed on wave III  and wave V (larger in CDCs).Wave amplitudes were typically below 20 iability . When coP = 0.9 for interpeak intervals II-III,  P = 0.86 for interpeak III–IV,  P = 0.17 for interpeak IV-V,  P = 0.14 for interpeak II–V).Wave I was difficult to evaluate in deaf cats, as the recordings did not include this peak in sufficient number of animals. This was attributed to a shorter latency of this wave in deaf animals (by which the wave and the stimulus artifact coincided). Latencies of waves II–V at 4 dB above threshold were not significantly different between deaf and control animals in this age group . Also alFor the first time the present manuscript describes development of brainstem-evoked responses in inborn deafness. The developmental pattern of brainstem evoked responses with well-controlled auditory stimulation (through a cochlear implant) was compared between hearing controls and congenitally deaf cats. Using electrical stimulation it has been possible to investigate brainstem-evoked response development prior to hearing onset. At postnatal day 3, E-ABRs could be evoked, corresponding to “acoustic” studies with first responses at postnatal day 2-3 using an unphysiologically high sound pressure level of 140 dB .The study demonstrates a developmental change in the brainstem evoked responses in both hearing and deaf animals. Thus, many functional developmental steps in the brainstem are set-in by genetic programs and do not require a functional cochlea. This stands in contrast to the findings in the auditory cortex, where the developmental pattern has been found extensively modified by deafness .However, several effects of deprivation were observed in the brainstem responses. Qualitatively, the E-ABR appeared more smeared and were more variable in CDCs and indicated a desynchronization of the underlying neuronal activity in CDCs. Quantitatively, there was an increase in thresholds of early E-ABR waves and divergent effects of deafness on waves II, III, and V with respect to amplitudes and thresholds. Wave V has generators in the midbrain, whereas earlier waves are generated in the brainstem , 25, 26.Considering that brainstem-evoked responses are not capable reflecting all details of the differences in synaptic and neuronal function between HCs and CDCs, the effects of deafness on waves II–IV are well in agreement with studies demonstrating synaptic reorganization in the cochlear nucleus following congenital deafness , 29–35, Several methodological factors could have influenced the present results. First of all, amplitudes of brainstem responses are known to have a higher variability than latencies (reviewed in ). This iThe threshold of E-ABRs depends on the position of the cochlear implant within the scala tympani. Positioning the stimulation electrodes closer to the modiolus can decrease E-ABR thresholds by 6 dB . The posThe number of surviving spiral ganglion cells may significantly influence threshold currents . NonetheFinally, the repetition rate could have influenced the results, especially in the youngest animals. To avoid confounding the results by different repetition rates in different age groups, we decided to select a constant low repetition rate (13 Hz), whereas the brainstem response in adults can be reliably recorded up to the repetition rates of 70 Hz . Even inElectrically evoked brainstem responses are similar to acoustically evoked brainstem responses –45. ConsDevelopment of brainstem responses to electrical stimulation has not been systematically investigated yet. On the other hand, development of brainstem-evoked responses to acoustical stimulation has been investigated in great detail in hearing cats comp. , 47, 48), 4847, 4The present experiments provide further support for the ABR hearing screening procedure performed on the deaf white cats : none ofElectrically evoked brainstem responses can be recorded even before acoustically evoked responses with physiological sound pressure levels. This demonstrates that the afferent auditory pathway (at least in part) is already functional before significant hearing is possible. Additionally, it shows that the general pattern in the afferent auditory pathway develops in absence of auditory experience Wave I corresponds to compound action potentials of the auditory nerve . In the The present results on matured animals are in accordance with data on neonatally pharmacologically deafened adult cats. Pharmacologically deafened adult animals show smaller E-ABR amplitudes : this waAnother mechanism for smaller amplitudes of the E-ABRs in deaf cats could be a loss of neurons in the cochlear nucleus of deprived animals. This was shown in rodents when the neuronal activity was completely silenced before hearing onset , 58. To Numerous studies described morphological changes in the synapses of the brainstem in CDCs , 32–35. Delayed or incomplete myelination in CDCs may be a further reason for smaller E-ABR amplitudes in CDCs. However, the absence of differences in interpeak latencies between CDCs and HCs argue against a delayed transmission along the auditory brainstem and thus also against this interpretation. Further studies are required to eventually identify the mechanism behind smaller E-ABR amplitudes in CDCs.There was an increase in individual wave II–IV thresholds in CDCs. This was also observed in adult neonatally deafened animals . IncreasDevelopment of human brainstem-evoked responses has been also described in great detail in hearing children (review in ). BrainsCorrespondingly, no sensitive periods in the development of brainstem response of humans have been observed with chronic electrical stimulation . ThElectrically evoked brainstem responses show postnatal development even in complete absence of hearing experience. Deprivation-induced effects include reductions of wave III amplitude, increase of wave V amplitude, and increases of wave thresholds. No effects of deafness on E-ABR latencies were found. The results indicate desynchronization and/or weakening of synaptic activity in auditory brainstem and some additional compensatory “hypersensitivity” in the midbrain of deaf animals.


Surgical site infection (SSI) after colorectal procedures represents a measurable quality indicator of a health care system. There is interest in comparing SSI rates between different hospitals and countries, however variability of the data regarding to incidence of SSI makes this comparison controversial. For the purposes of evaluation, data must be standardized and include reliable post-discharge surveillance (PDS).To determine rates of SSI after elective colorectal procedures among hospitals of the VINCat Program.VINCat is a nosocomial infection surveillance program in Catalonia, Spain. Between 2007 and 2012, 59 hospitals joined the program. The participating hospitals performed active, prospective, standardized surveillance of elective colorectal resection. PDS was implemented by a multimodal approach and was mandatory within the first 30 days after surgery. Since 2011 colon and rectal procedures were also analysed separately.During the study period, 17,779 elective colorectal procedures were included. Mean age was 69y (SD:12y) and 40% were female. SSI was diagnosed in 3,485 (20.3%) patients. Among them, 782 (22.4%) were diagnosed during PDS. Median time from surgery to infection was seven days (IQR 5-9) for in-hospital SSI and 14 days (IQR 10-19) for PDS-SSI. Surgical infections due to colon procedures were only slightly lower (18.8%) than those due to rectal surgery (22.3%). Both, overall SSI rates and organ/space SSI rates did not change significantly over the study period and were respectively: 2007 (20.8%/5.3%), 2008 (19.2%/6.9%), 2009 (21%/9%), 2010 (21%/8.5%), 2011 (20.7%/9.3%) and 2012 (19%/8.9%).SSI rates in elective colorectal procedures at VINCat hospitals remained stable over the study period and were higher than those reported by other national programs. There is a need to clarify what surgical site infection rates in colorectal surgery should be considered for benchmarking standards.None declared.


Our data suggest that sulfated GAGs play a major role in EDN binding, which in turn may be related to the cellular effects of EDN.Human eosinophil derived neurotoxin (EDN), a granule protein secreted by activated eosinophils, is a biomarker for asthma in children. EDN belongs to the human RNase A superfamily possessing both ribonucleolytic and antiviral activities. EDN interacts with heparin oligosaccharides and heparin sulfate proteoglycans on bronchial epithelial Beas-2B cells. In this study, we demonstrate that the binding of EDN to cells requires cell surface glycosaminoglycans (GAGs), and the binding strength between EDN and GAGs depends on the sulfation levels of GAGs. Furthermore,  It is a.9 normalza virus . Furtherza virus  and amyoza virus .EDN and its mouse counterpart, mouse eosinophil-associated RNase 2 (mEAR2), have been reported to act as a selective chemoattractant for dendritic cells (DCs) . They prin vitro experiments, but also heparan sulfate (HS) expressed on the surface of Beas-2B cells. Heparin and HS are linear polysaccharides consisting of repeating disaccharide units of α-1,4-linked hexuronic acid and hexosamine [O-sulfated IdoA and 6-O- and N-sulfated GlcN [EDN shows affinity for heparin, as indicated by its purification in 1986 using heparin-Sepharose column chromatography . We havexosamine . The hexxosamine . Most coted GlcN . In addited GlcN . These Gted GlcN ,25.34QRRCKN39 in loop 3, 65NKTRKN70 in loop 4, and 113NRDQRRD119 in loop 7 [34QRRCKN39 matches exactly to the XBBXBX motif [32NYQRRCKNQN41, has been demonstrated to be capable of binding heparin [65NKTRKN70 also possesses the XBBXBX pattern in a reverse order, but 113NRDQRRD119 does not have any known heparin binding sequence. To date, the second and the third regions serving as binding sites for heparin in EDN have not been described.It is empirically known that heparin binding proteins have domains characterized by the presence of clusters of positively charged residues, such as Arg and Lys, which are likely to promote heparin binding by electrostatic interactions . Two conn loop 7 . All of BX motif , and ind heparin . Regardi34QRRCKN39, 65NKTRKN70 and 113NRDQRRD119 were identified as heparin binding regions (HBRs)—i.e., HBR1, HBR2 and HBR3, respectively—and their functional roles in heparin binding were characterized using in silico computer modeling and in vitro binding assays. Furthermore, the importance of sulfo groups of GAGs in interaction with EDN was characterized.In this study, the sequences et al. [N-terminus. Nevertheless, potential influence of bulky MBP tag in EDN function by blocking GAG binding sites would be further evaluated.Neuton D. L. et al.  have expvs. AMAC-LMWH, only 10% MBP appeared as a complex with AMAC-LMWH. Concentration-dependent binding of MBP-EDN to Beas-2B cells was also observed by cELISA. MBP showed only a background level of signals (11%), compared to that of MBP-EDN (100%) at 0.8 μM (Binding of recombinant MBP-EDN to heparin and Beas-2B cells was characterized by FACE and cELISA . In the t 0.8 μM . These dTo further differentiate differences in GAG binding affinity between MBP-EDN and refolded recombinant EDN (EDN-6His), heparin binding activity of both was measure as shown in Interaction of MBP-EDN with cell surface GAGs was further investigated using wild type Chinese hamster ovary (CHO) cell line (CHO-K1) , and twoIn the mutant CHO cell line pgsD-677, synthesis of HS is specifically impaired due to lack of HS polymerase that polymerizes disaccharide units of outer HS chains. However, pgsD-677 cells have approximately three times more CS as compared to CHO-K1 cells . The amoTo further investigate specificity of MBP-EDN binding, we measured the inhibitory activity of different types of GAGs including high molecular weight heparin (HMWH) which is highly sulfated, chondroitin sulfate C (CSC) and dermatan sulfate (DS) which are moderately sulfated compared to HMWH, and hyaluronic acid (HA). Among these four GAGs, HMWH showed a high level of inhibition (87%) compared with the binding of LMWH to MBP-EDN, and DS reduced LMWH binding by 50% at a GAG: LMWH ratio of 5. However, the same amount of CSC and HA had no effect . SimilarN-acetylgalactosamine , and is distinguishable from chondroitin sulfate A (CSA) (4-O-sulfated) and CSC (6-O-sulfated) mainly in the presence of IdoA moiety which also appears in heparin/HS [N in common, in contrast to GalN in DS and CSC. However, the inhibitory activity of HA was very low. These data discount the possibility of the GlcN moiety being involved in EDN-GAG interactions.HMWH, the most sulfated GAG containing on average 2.7 sulfo groups per disaccharide unit, possessed higher inhibitory activity than DS and CSC that have one sulfo group per disaccharide unit as expected . Howeverparin/HS . Since tparin/HS , IdoA moparin/HS . Cell suparin/HS . These rMany biological activities attributable to EDN and ECP have been characterized and found to be significantly different; nevertheless, EDN and ECP still present similar properties such as ribonucleolytic , antivirN-acetyl heparin (NAcH), de-N-sulfated heparin (deN-SH), N-acetyl-de-O-sulfated heparin (NAc-deO-SH), de-2-O-sulfated heparin (de2O-SH), and de-6-O-sulfated heparin (de6O-SH). NAc-deO-SH (completely devoid of O-sulfo group and with a markedly diminished N-sulfation) showed the weakest inhibition (2%) of MBP-EDN binding to AMAC-LMWH, indicating that the sulfo groups provided almost all necessary negative charges for binding to EDN. Four other derivatives with selectively modified sulfo groups including NAcH, deN-SH, de2O-SH, and de6O-SH showed 31%, 8%, 43%, and 39% inhibition activities, respectively. Similarly, results of cELISA competition assay revealed that HMWH inhibited 87% of cellular binding of MBP-EDN while NAc-deO-SH showed only 7% inhibitory effect at a concentration of 10 μg/mL. Regarding other derivatives, deN-SH, NAcH, de2O-SH, and de6O-SH showed 34%, 69%, 66% and 70% inhibition activities at the same concentration, respectively, compared with the control without competitor. At 50 μg/mL, all heparin derivatives showed more than 50% inhibition whereas NAc-deO-SH inhibited only 24%  signaling  and viruonly 24% .O- or 6-O-sulfo groups showed a decreased MBP-EDN binding activity, it could not completely abolish the interaction between MBP-EDN and Beas-2B cells. This suggests that EDN might not interact with heparin through only one specific position of sulfo group, unlike FGF-1 or FGF-2, which require 6-O sulfated [O sulfated heparin [O-sulfo group which rarely appears in heparin/HS and has been proposed to be important for interaction with FGF-7 [N-sulfo groups, although both NAcH and de-N-SH were able to significantly reduce MBP-EDN binding to Beas-2B cells, the inhibitory effect of NAcH (69%) was higher than that of de-N-SH (34%). Besides, out of these two derivatives, only NAcH was observed to compete with the interaction between LMWH and MBP-EDN by FACE analysis, suggesting that the N-acetyl group of heparin, which appears in most HS sequences, may be responsible for weaker binding to MBP-EDN.Although heparin lacking of 2-sulfated  and 2-O  heparin  for inteth FGF-7  and infeth FGF-7  might al36 and Lys38 in HBR1, and Lys1 and Arg132 which did not belong to HBRs, whereas Arg97 and basic residues in HBR2 and HBR3 were not located within van der Waals force/hydrogen bond distances to interact with the heparin hexasaccharide  and 113NRDQRRD119 (HBR3) play auxiliary roles in the interaction between MBP-EDN and GAGs. Natural heparin is a heterogeneous mixture with molecular weights ranging from 5000 Da to over 40,000 Da (HMWH). LMWHs, in contrast, consist of only short chains of heparin of average molecular weight of less than 8000 Da (approximately 10 disaccharide units). The basic residues of HBR2 and HBR3 which are too far to interact with the heparin hexasaccharide in the computer model might act as a secondary heparin binding sites on EDN and bind with much lower affinity to long chain heparin molecules. Recently, the loop L7 of EDN containing HBR3 has been shown to play an important role in anti-hRSV activity of EDN [MBP-EDN-HBR mutants, in which the basic residues in HBRs were replaced with Ala, were generated to investigate the functional roles of HBRs in EDN. All mutants showed a weaker interaction with AMAC-LMWH at different molar ratios of protein/GAG . The int MBP-EDN . This suy of EDN . Since hy of EDN , EDN mig34RWRCK38, 73RSRFR77, and 101RPGRR105 have been aligned with the corresponding segments of the other 12 RNases employing Clustal W2 [34RWRCK38) is found in a position comparable with HBR1 on EDN (34QRRCK38) within a primary sequence that is 60% homologous. However, all the HBRs on EDN and ECP were located at a similar position and surrounded by an electropositive cavity in their tertiary structures. Peptide containing HBR1 in ECP (NYRWRCKNQNK) had similar LMWH binding activity to the corresponding region of EDN (NYQRRCKNQNK) in FACE analysis [1, Arg7 and His64) and polar residues  surrounded in a basic cavity as prediction model illustrated in a previous study [Putative HBRs on ECP including ustal W2 . Among tanalysis . Howeveranalysis . Here, mus study .66 and Arg68 resulted in little or insignificant effects  showed major contribution in cellular binding, it still possessed 45% binding activity to LMWH in FACE analysis. In addition, there are six hydrogen bonds in our EDN-heparin hexasaccharide prediction model, revealing that contribution of other aromatic and polar residues such as His, Trp, Asn and Gln should be taken into consideration.ECP structure in complex with heparin disaccharide  and trisN-acetyl heparin (NAcH), de-N-sulfated heparin (deN-SH), and N-acetyl-de-O-sulfated heparin (NAc-deO-SH) were purchased from Sigma-Aldrich . De-2-O-sulfated heparin (de2O-SH), and de-6-O-sulfated heparin (de6O-SH) were purchased from Neoparin . Chemicals were purchased from Sigma-Aldrich  unless otherwise specified.Monoclonal mouse anti-MBP was purchased from Santa Cruz  and goat anti-mouse horseradish peroxidase (HRP) was purchased from Jackson ImmunoResearch . Low molecular weight heparin (LMWH), high molecular weight heparin , dermatan sulfate (DS), chondroitin sulfate C (CSC), hyaluronic acid (HA), v/v) fetal bovine serum (FBS)  and 1% (v/v) Penicillin, Streptomycin, and Amphotericin . CHO cells were cultured in Vitacel Ham’s F12K medium  supplemented with 10% (v/v) FBS.Beas-2B (ATCC number: CRL-9609), a human bronchial epithelial cell line, wild type Chinese hamster ovary (CHO)-K1 (ATCC number: CRL-9618), mutant CHO cell lines pgsD-677 (ATCC number: CRL-2244) and pgsA-745 (ATCC number: CRL-2242) were purchased from American Type Culture Collection . Beas-2B cells (ATCC number: CRL-9609) were cultured in RPMI 1640 medium  supplemented with heat-inactivated 10% . In addition, these residues were simultaneously substituted to Ala to generate the following three mutants: MBP-EDN-HBRmt1 (R35A/R36A/K38A), MBP-EDN-HBRmt2 (K66A/R68A/K69A), and MBP-EDN-HBRmt3 (R114A/R117A/R118A) using the same protocol. To express a wild type MBP-EDN and its mutants, the plasmid was used to transform Escherichia coli (E. coli) BL21 (DE3) Gold cells . Two milliliters of the overnight culture was inoculated in 100 mL LB broth containing 100 μg/mL carbencillin, and incubated at 37 °C until OD600 reached 0.4–0.6. IPTG  was added to a final concentration of 0.5 mM, and incubated at 30 °C for 4 h. After protein induction, the bacteria were harvested by centrifugation for 10 min at 2900× g, and the pellet was homogenized by EmulsiFlex-C3 . Wild type MBP-EDN and all mutant proteins in the soluble portion of bacterial cell lysates were purified by amylose affinity chromatography .The DNA encoding EDN without the signal sequence was amplified using PCR primers 5′-GAATTCAAACCTCCACAGTTTACC-3′ and 5′-TCTAGATTAGATGATTCTATCCAG-3′ and cloned into the pMAL-c2G plasmid  between the edn gene was cloned into the pET23a plasmid  between XbaI and BamHI to generate pET23a-edn. The expression procedures were same as MBP-EDN but misfolded EDN-6His was accumulated in inclusion bodies. Recombinant EDN-6His was purified by Ni2+ Sepharose™  and refolded and dialyzed as previously described [The escribed .® West Pico Chemiluminescent Substrate  was added and the chemiluminescent intensity was measured by Wallace Vector II . The amount of MBP-EDN bound to cells without heparin derivatives or GAGs treatment was set to 100%.Confluent monolayers of Beas-2B or CHO cells in 96-well plate were incubated with different concentrations of MBP-EDN at 4 °C for 60 min. The cells were washed with 200 μL of ice-cold PBS, and fixed with 2% paraformaldehyde (PFA)/PBS at 25 °C for 15 min. Then, 200 μL of ice-cold PBS was added to wash the cells prior to blocking with 2% BSA/PBS at 25 °C for 90 min. The level of bound MBP-EDN was quantified by ELISA analysis using a mouse monoclonal anti-MBP IgG and a HRP-conjugated goat anti-mouse IgG as the primary and secondary antibody, respectively. After the cells were washed by ice-cold PBS, 50 μL of Super Signal3CN) was added, and the mixture was incubated at 37 °C for 16 h. At the end of the reaction, 720 μL of 95% ice-cold ethanol was added and rested for 15 min at −20 °C. The sample was centrifuged at 4 °C for 5 min at 14,000× g. The supernatant was carefully discarded, and the pellet was freeze-dried by ScanVac CoolSafe . The dried pellet was dissolved in sterile deionized water at an appropriate concentration for a measurement. The AMAC-labeled probes thus prepared were stored at −80 °C until use.GAGs were labeled with 2-aminoacridone (AMAC) according to the reported procedures . BrieflyAMAC-labeled probes and proteins were mixed and incubated at 25 °C for 15 min. The mixture was loaded onto a 1% agarose gel plate, and electrophoresed for 20 min in a buffer containing 40 mM Tris-acetic acid, 1 mM EDTA, pH 8.0. The AMAC labeled probe was detected under UV light and the gel was scanned by QUANTUM-ST4 .For competition assay, monolayers of Beas-2B cells in 96-well plates were pre-incubated with serially diluted heparin derivatives or GAGs in serum-free RPMI 1640 medium at 4 °C for 30 min. To the samples, MBP-EDN was added, and incubated at 4 °C for 60 min. Bound MBP-EDN was measured as described above.In silico docking experiments were performed using AutoDock Vina software  [1C4-IdoA(2S)–GlcNS(6S). In order to find preferred side chain conformations, all basic residues in three HBRs of EDN were set to be flexible in computation. Global search was performed in the grid box with a dimension of 60 × 60 × 60 angstroms that covered the entire protein. For computer simulation, we chose heparin hexasaccharide with chair forms of IdoAs as a ligand and used predetermined coordinates for the side chains of basic residues within HBRs.The structure of EDN was taken from PDB database (PDB entry 1HI2) , and eneCA, USA) . Prelimit-test.Statistical analysis was performed using GraphPad Prism 5 . All data are shown in mean ± standard deviation (SD). Statistical significance between a set of data was determined by unpaired two-tailed Student’s O- or 6-O-sulfo group, is an obligatory factor for EDN interaction to heparin and Beas-2B cells. A combination of in silico bioinformatics analyses and in vitro functional assays has successfully identified that basic residues Arg35, Arg36, and Arg38 in HBR1, and Arg114 and Arg117 in HBR3 of EDN play a critical role in GAG binding. These results provide an insight into the molecular interaction of EDN with soluble heparin and bronchial epithelial cells which may have biomedical implications for the physiological functions of eosinophil RNases.In conclusion, membrane GAGs, especially HS and DS, were essential for EDN interaction with Beas-2B cells. The Sulfo group of heparin, predominantly 2-


Horizontal gene transfer (HGT) is a process that facilitates the transfer of genetic material between organisms that are not directly related, and thus can affect both the rate of evolution and emergence of traits. Recent phylogenetic studies reveal HGT events are likely ubiquitous in the Tree of Life. However, our knowledge of HGT's role in evolution and biological organization is very limited, mainly due to the lack of ancestral evolutionary signatures and the difficulty to observe complex evolutionary dynamics in a laboratory setting. Here, we utilize a multi-scale microbial evolution model to comprehensively study the effect of HGT on the evolution of complex traits and organization of gene regulatory networks.Large-scale simulations reveal a distinct signature of the Distribution of Fitness Effect (DFE) for HGT events: during evolution, while mutation fitness effects become more negative and neutral, HGT events result in a balanced effect distribution. In either case, lethal events are significantly decreased during evolution (33.0% to 3.2%), a clear indication of mutational robustness. Interestingly, evolution was accelerated when populations were exposed to correlated environments of increasing complexity, especially in the presence of HGT, a phenomenon that warrants further investigation. High HGT rates were found to be disruptive, while the average transferred fragment size was linked to functional module size in the underlying biological network. Network analysis reveals that HGT results in larger regulatory networks, but with the same sparsity level as those evolved in its absence. Observed phenotypic variability and co-existing solutions were traced to individual gain/loss of function events, while subsequent re-wiring after fragment integration was necessary for complex traits to emerge. Horizontal Gene Transfer (HGT) is the transport of genetic material within and across species. It is a mechanism of genetic exchange complementary to vertical transfer, which occurs through cell division and results in the transfer of genetic information from an ancestor to its offspring cells. Although largely ignored in the past, recent phylogenetic evidence suggests that its impact on bacterial evolution is significant and should be investigated more thoroughly ,2. For iDue to our limited ability to observe HGT dynamics in an experimental setting, theoretical models have been traditionally employed to elucidate the impact of HGT on evolution. Continuous ,8 and stin silico organism encompass functions and parameters that model basic biological phenomena, while its core consists of a gene regulatory and biochemical network with abstract molecular representations. Instead of imposing an arbitrary, artificial selection function  as in previous studies, we instead created an environmental model that includes nutrients. The model has been extended to incorporate the Horizontal Gene Transfer in addition to other cellular  and evolutionary  processes previously included in the model . Additihods and  to handlAn overview of the simulation setting discussed in this paper is illustrated in Figure 1 sand 2 scarry information regarding the presence of nutrients in the environment . Similarly, the correlation-structure of environments A and B is Nutrients Presence [A] = Delayed (1 sAND NOT(2s)) and Nutrients Presence [B] = Delayed (NOT(1s) AND 2s), respectively. The introduction of a delay in the signal/nutrient correlation further increases the evolutionary complexity of the environment, as organisms now have to account for it, through the underlying network topology and dynamics. In the absence of delays between nutrient-signal occurrences, we obtained the same observations, but evolution took place faster, and networks tended to be less complex. The three environments were not randomly selected: despite the fact that the combined AB environment (delayed XOR) is a simple combination of the A and B environments, its complexity is significantly higher when compared to the other two (A and B). The main reason behind the increased complexity of environment AB is the fact that is not linearly separable ; i s∈ [1-4]; i basalis ∈; initial sparsity of the regulatory network is ; connection to the input signals of each additional node is proportional to exp(-n), where n is the number of nodes already regulated by input signals; initial energy of the organism 800,000 energy units; size of the initial random network can be up to 15 triplets.Initial unevolved populations are composed from randomly generated cells with the following parameters: There are three mechanisms for HGT by which bacteria can acquire external DNA: transformation, conjugation and transduction (e.g. review ), which -7 and 10-11 per generation, per cell [-3 to 10-1 [0 and triplet T0 by the transferred sub-network is preserved  of HGT events is in the upper range of the experimentally observed values, and consistent with the rest biological and evolutionary model.Experimentally observed HGT rates between bacteria in natural environments vary between 10per cell -24, whil to 10-1 . In our Distribution of the fragment sizes in HGT events may vary greatly in the bacterial world and depends on the type of the transfer and experimental conditions. However in all three types of HGT the maximum size of the transferred DNA is limited by different parameters: in transduction by the capacity of the viral capsid, in conjugation by the time two organisms stay connected by a pilus, and in transformation by the stability of the naked DNA in the environment. In general the probability of transfer small fragments of meaningful DNA is higher than of larger ones. In our model triplets with preserved regulatory network are transferred from one organism to another, and the fragment size for an established HGT event is chosen using a probability density function as a normalized sigmoid function:n is the fragment size in triplets, m and s are the middle point and slope of the probability density function, respectively; the denominator is a normalization coefficient. In most cases s = m was used throughout the paper, and therefore 67% of all transferred fragments were not larger than m triplets . Unless otherwise mentioned, the default parameters for HGT in environments tested in our paper were set to (s = m = 5) which results in the expectation value for the size of HGT fragments equal to 4 triplets and slightly smaller than the average size of the minimal network  with a near-linear speedup for a load of 8 cells per core. At every time step organisms mutate with predefined probabilities and node values are updated using the stochastic expression model described above; cells which exhausted their energy are removed from the population and replaced with new random cells (to start from a new point on the fitness landscape); cells which reach an energy above the division threshold are duplicated, and daughter cells replace cell with low energies to maintain a constant population size.modus operandi of each evolved network, which may have hundreds of nodes and links, we developed the following heuristic to reduce the network to its "minimal" form, in which only essential nodes and links remain. In this iterative procedure, the fitness effect of a link is assessed after its severance. The link is permanently removed if it is deemed non-essential (less than 5% fitness change). The procedure is repeated until the network cannot be reduced any further. Due to the stochastic nature of the expression model, fitness of a cell can vary as much as 30% between sequential epochs. For that reason the average fitness is evaluated over 10 epochs to reduce that variation to 2%. Multiples iterations over all edges with a tight removal threshold ensure gradual and stable reduction on the network to a near-optimal minimal sub-network.To elucidate the 0+dz)/w(z0)w w(zand 0+dz) w; kurtosis is the measure of 'peakedness' (positive if distribution is 'sharper' then a Gaussian distribution).Mean, variation, skewness and kurtosis for several experimentally measured DFEs are summarized in Table To assess the effect of HGT in originally non-evolved populations, we placed random populations in all three environments  with and without the presence of HGT. In the absence of pre-evolved cells or even partial solutions, the presence of HGT was found not to significantly alter the fitness trajectories of the corresponding populations. As shown in Figure XOR function , only 18 of 32 (56%) experiments were successful and terminated with an evolved XOR population . In contrast, in the case of dual-step adaptation process, where populations where first introduced to environment A or B and then to the AB environment, the success rate was considerably higher to 94% and 85%, with and without HGT, respectively. In addition, HGT accelerates by a factor of 1.7 the emergence of the delayed XOR phenotype in {A, B} mixed populations . This HGT effect is less pronounced, however, at the stage of phenotypic refinement (i.e. fitness levels above 0.9) as it is mutations, rather than the integration of new functional fragments (i.e. newly-transferred sets of triplets), that are responsible for fine-tuning of the expression dynamics.Detailed statistics of the evolution success rate and speed are shown in Tables -5 . The rate of evolution steadily increases with the increase of the average fragment size transferred by HGT, and saturates at approximately 5 triplets. A closer look at the underlying networks provides the reason behind this saturation limit. In the AB environment the average network size of the evolved organism is 9 triplets, however the average network size that is essential to exhibit the delayed XOR phenotype is only 5.5 triplets . The presence of HGT clearly leads to a faster emergence of fit phenotypes (30 epochs vs. 150 epochs). In addition, we looked at how the rate of evolution scales as a function of the population size. It is believed that evolution speed increases linearly with population size N for small populations, and with ln(N) for intermediate population sizes [9) [In the presence of HGT and in combined populations, the on sizes , while iizes [9) . In our A and B, along with their offspring, were found to be replaced within 50 epochs by the evolved AB (XOR) phenotype, once the latter arises. However, both phenotypes A and B persisted until the end of the simulation runs and at relatively high percentages  when introducing mutations in a population. These distributions have been measured experimentally for viruses and bacteria -37 and hectively ,32.Theoretical approximations of the DFE are usually based on the concept that most mutations are "nearly neutral" as introduced by Kimura . Followiin silico simulation framework to investigate the shape and changes in the DFE for both mutations and HGT. Since each organism has its own regulatory network that results to a distinct phenotypic behavior, we are able to calculate fitness before and immediately after any HGT event (correlation between expression levels of the response pathway and nutrient presence). This allows us to profile the shape of DFE along the evolutionary trajectory and to account for genetic drift, which can be a significant force in small populations. Figure w > 0.75) sampled from populations that had evolved in an XOR environment for 8,000 epochs. In both mutation and HGT DFEs, there is a profound decrease in the number of lethal events  in evolved populations versus the non-evolved populations, a clear indication that the latter become robust to mutations. Table E. coli. Furthermore, we observe a decrease in variance and increase in kurtosis (sharpness) of the DFE as populations evolve, both for HGT and mutations, although the effect is more profound in the latter case. As population evolves, mutation DFE becomes more skewed towards negative fitness effects, which is to be expected as most mutations in an evolved organism result in a decreased fitness. Interestingly, the DFE of HGT events becomes more symmetric (skewness closer to zero) in evolved populations, as the probability for HGT to transfer a beneficial or disrupting fragment increases .Here, we use our To gain more insight on the DFE changes, we traced the fitness effect of mutations and HGT events along a single evolutionary trajectory. Figure vs. 0.22), but with the same average sparsity and reduced network size difference when it comes to their minimal counterparts.The complete gene regulatory and biochemical network of an evolved cell is usually too complex to analyze, while many of its connections are not relevant to the observed phenotype. To address this issue, we employed a reduction algorithm (see Methods) to extract the "minimal" network that encompasses only essential connections. As shown in Table w = 0.84 in the AB environment (XOR) which is unusually high for the given timeframe. The expression profile of the donor, acceptor and final cell is depicted in Figure 1. The resulting cell is able to metabolize the nutrients during both pulses, and its response expression profile correlates well with the nutrient presence. Subsequent mutations result in the fine-tuning of the timing for the expression of the response pathway, which then correlates better to the second nutrient pulse (data not shown).To elucidate how HGT confers a fitness advantage during evolution, we dissect the gene regulatory and biochemical network of a cell before and after an HGT event. Figure Delayed (1 sAND NOT(2s))) being in a solid yellow background. Activation of the metabolic (response) pathway RP0 of triplet T0 is regulated by the protein from the triplet T2: signal S1 activates transcription of T3, protein T3 controls translation of T1, which in turn regulates the transcription of T2. The delay is introduced by activation of the protein modification in T2 by the same signal S1, which decreases concentration of un-modified protein T2 while signal S1 is present. Once signal S1 is off, modified proteins T2 gradually return to the un-modified state and metabolic pathway RP0 becomes activated. Signal S2 regulates protein modification in T3, which blocks metabolic pathway RP0 expression when both signals S1 and S2 are present, and therefore satisfies NOT(2s) part of the environment. The HGT event introduces three extra triplets that add the additional functionality as follows: signal S2 activates transcription of T5, which in turn activates the metabolic pathway RP0. Signal S1 activates transcription of T4 and T6, which inhibit translation of T5. Triplet T6 has parameters almost identical to T4, since it was created by triplet duplication earlier during the evolution. However in this cell, both T4 and T6 are present in the minimal network to maintain a required level of inhibition.Figure Horizontal gene transfer is a phenomenon that inarguably affects the evolution and emergence of complex traits. To what extent it does so, its impact to the underlying networks and population dynamics, however, is still to be determined. Our multi-scale models and simulations allow, for the first time, to address questions related to HGT that transcend three levels of organization , shedding light on its effect during evolution and providing concrete examples of network integration and operation. Our results show that the effect of HGT is very much dependent on the environmental context which includes, but is not limited to, the genotypic and phenotypic variability of the existing populations. The observation that HGT can accelerate the rate of evolution of heterogeneous populations is intriguing: can we experimentally manipulate the rate of evolution by sequentially exposing a population to fluctuating environments in the presence of HGT? What are the principles and rules that optimize this effect? Our computational results show that adaptation to correlated environments of increasing complexity can accelerate the rate of evolution, especially in the presence of HGT, a hypothesis that remains to be tested in a laboratory setting. HGT events show a distinct spectrum in respect to their fitness effect. The HGT-derived fitness effect distribution was found to increase its symmetry during evolution, in contrast to the fitness effect distribution of mutations, which becomes more skewed towards deleterious mutations. Our distributions of mutation effects are in complete agreement with experimental observations so far, while our predictions regarding the reported DFE bias remain to be tested experimentally.a posteriori analyze the effect of every and each mutation event during the evolution. The proposed framework can serve as a predictive and testing tool for evolutionary hypotheses that would be difficult to evaluate experimentally, while at the same time can drive laboratory experimentation by providing scenarios that are likely to occur in specific environmental contexts.There are many future directions to explore in order to increase the scope of the methods presented here. In respect to the models, the current framework will benefit from the addition of a spatial component, since in the current setting we assumed a well-mixed, homogeneous environment which doesn't allow the investigation of individual HGT mechanisms , as their effect varies greatly with the spatial landscape of the environment and population structure. Moreover, the same simulations can be performed with a model of adaptively growing populations which are only nutrient-limited instead of having a fixed-size population, although the latter is easier to analyze and balance in simulations. A larger population size is always desired as small effective population size renders results prone to undesired artifacts, such as random drift, although this is less of a concern here as we can DFE: distribution of fitness effect; HGT: horizontal gene transfer.The authors declare that they have no competing interests.Corresponding author IT conceived the project; VM wrote the code and performed the experiments; VM and IT designed the methods, analyzed the data and wrote the manuscript.Evolution rate as a function of the population size. Rate is calculated as an average slope of the maximum fitness increase averaged over 8 independent experiments for each population size of 256, 512, 1024, 2048, 3072 and 4096. Initial random populations evolved in the XOR environment until the maximum fitness is stabilized.Click here for fileHGT fragment transfer probability and genome integration. (A) Probability density function profile used to select HGT fragment sizes. (B) Incorporation of the HGT-transferred fragment (triplets TN+1 to TN+k) in the regulatory matrix, where only the response pathway (triplet T0) regulation is conserved. The newly acquired fragment can over time rewire and couple to other nodes in the network. Simulations where the imported fragment was randomly rewired to the host genome yielded similar results.Click here for file


Cordyceps bassiana according to fruiting body developmental stage were investigated using gas chromatography-mass spectrometry. We were able to detect 62 metabolites, including 48 metabolites from 70% methanol extracts and 14 metabolites from 100% n-hexane extracts. These metabolites were classified as alcohols, amino acids, organic acids, phosphoric acids, purine nucleosides and bases, sugars, saturated fatty acids, unsaturated fatty acids, or fatty amides. Significant changes in metabolite levels were found according to developmental stage. Relative levels of amino acids, purine nucleosides, and sugars were higher in development stage 3 than in the other stages. Among the amino acids, valine, isoleucine, lysine, histidine, glutamine, and aspartic acid, which are associated with ABC transporters and aminoacyl-tRNA biosynthesis, also showed higher levels in stage 3 samples. The free radical scavenging activities, which were significantly higher in stage 3 than in the other stages, showed a positive correlation with purine nucleoside metabolites such as adenosine, guanosine, and inosine. These results not only show metabolic profiles, but also suggest the metabolic pathways associated with fruiting body development stages in cultivated C. bassiana.The metabolic profiles of  Cordyceps bassiana, belonging to the Ascomycetes, is known to be an entomopathogenic fungus for insects and other arthropod pests with a broad host range, and C. bassiana was found growing in a Lepidopteran cadaver in the wild C. bassiana was recently successfully cultured in vitro for commercial use C. bassianaFungi are used extensively in the production of a broad range of metabolites, such as pharmaceuticals, enzymes, and toxins for plants, animals, and humans Penicillium using mass spectrometry AspergillusC. bassiana during development using nuclear magnetic resonance spectrometry has recently been reported C. bassiana have not yet been elucidated. Moreover, most studies on Cordyceps species during fruiting body formation have only focused on targeted chemical composition change A metabolomic approach has been used for major metabolite identification in fungus as well as for fungal taxonomy of Arabidopsis, and a global test for metabolic pathways in Escherichia coli and Saccharomyces cerevisiae under different conditions have been reported by functional enrichment analysis The enrichment analysis method was recently developed for the functional interpretation of large amounts of data in the fields of genomics, transcriptomics, proteomics, and metabolomics C. bassiana according to fruiting bodies, which are classified from stages 1 to 4 during the formation of stromata and perithecium of fruiting body surfaces . Perithecia that from the stromata are flask-shaped structures containing ascospores, and the characteristic morphology of C. bassiana stromata has been reported previously C. bassiana fruiting bodies were categorized by the degree of perithecium formation in this study.However, to the best of our knowledge, no research using enrichment analysis has investigated metabolite annotation or metabolism changes in C. bassiana. In this study, we performed metabolic profiling of cultivated C. bassiana in various developmental stages using gas chromatography–mass spectrometry (GC-MS). In addition, the free-radical scavenging activities of those samples and their correlation with specific metabolites were investigated. The main objectives of this study were metabolic profiling and investigation of the free-radical scavenging activities in cultivated C. bassiana at various developmental stages. The major metabolic pathways associated with developmental stages will also be discussed.The main hypothesis is that the metabolite level associated with specific metabolisms and free-radical scavenging activity might change according to developmental stage of cultivated C. bassiana is B. bassiana, and the nuclear intergenic region Bloc and the nuclear ribosomal internal transcribed spacer region (ITS) were used to confirm the isolates used for fruiting body development in this study were C. bassiana based on the recent phylogenetic analyses of Rehner et al C. bassiana strains were grown on Sabouraud dextrose +1% (w/v) yeast extract broth (SDY) for 3 days at 25°C as inocula for the production of fruiting bodies of C. bassiana. The cultures of fruiting body were grown in 1,000-ml plastic bottles containing brown rice medium and incubated at 20°C under light intensity of 400 lux and 90% humidity. Samples were collected every week from the fourth to seventh weeks of cultivation to monitor and compare the development of stromata and perithecia. Observations were made using a dissecting microscope  and the four developmental stages were classified as follows: stage 1, week 4 (no perithecia formation on any stroma); stage 2, week 5 ; stage 3, week 6 ; stage 4, week 7 . The fruiting bodies isolated from culture media were freeze-dried and powdered according to developmental stage. Samples were stored at −70°C before analysis. Voucher specimens were deposited at the College of Pharmacy, Chung-Ang University, Republic of Korea (fruiting bodies: CAUCBF 20110904–20110907).The anamorph of n-hexane. After sonication, the tube was centrifuged at 2,000 rpm for 10 min. The supernatant was collected separately from each sample and filtered through a 0.45-um filter . After extraction, 100 µL of each sample solution was separately transferred into GC vials and then dried with nitrogen gas flow for 5 min at 60°C. After drying, 30 µL of 20,000 µg/mL methoxylamine hydrochloride in pyridine was added. Thereafter, 50 µL of BSTFA  trifluoroacetamide; Alfa Aesar, Ward Hill, Massachusetts) containing 1% TMCS (trimethyl chlorosilane) and 10 µL of 2-chloronaphthalene  were added to the dried sample. Derivatized samples were incubated at 60°C for 60 min, after which the solutions were directly used for GC-MS analysis.For quantification of purines  in C. bassiana samples, standard solutions (1–100 µg/mL) and sample solution  were prepared with 70% methanol. The sample and each standard solution of 90 µL were transferred into GC vial that was dried with nitrogen gas for 5 min at 60°C.The derivatization was performed as above described method. After derivatization process, the solution was directly used for GC-MS analysis. Characteristic ions of adenosine (230 m/z), guanosine (324 m/z), inosine (217 m/z), and uric acid (411 m/z) were selected in preliminary analysis, and those were used for each purine quantification of C. bassiana fruiting bodies.Methanol and n-hexane were used as solvents for the extraction of polar and nonpolar metabolites, respectively. Twenty milligrams of each sample in different growth conditions were transferred into glass Eppendorf tubes  and extracted with 1 mL of 70% methanol and 100% Samples were analyzed using a model 7890A Agilent GC  equipped with a model 5975C MSD detector (Agilent Technologies), an autosampler , a split/splitless injector, an injection module, and Chemstation software. The GC inlet temperature was set to 250°C with an injection volume of 1.0 µL and a split ratio of 1∶10, using helium as a carrier gas in constant-flow mode of 1.0 mL/min. A fused silica capillary column of 5% phenyl methylpolysiloxane phase  with dimensions 30 m×0.25 mm i.d. ×0.25 µm film thickness was used for analysis. The detector voltage was set to 1518 V, the auxiliary temperature was set to 280°C, the MS source temperature was set to 230°C, and the MS quad temperature was set to 150°C. The mass range was 50–700 Da. Data were obtained in full scan mode. The oven temperature for polar metabolite analysis was 80°C (hold 3 min) programmed to 130°C (3°C/min) then to 240°C (5°C/min) then to 320°C . For the non-polar metabolite analysis, the detector voltage was set to 1588 V, and the mass range was 50–600 Da. The oven temperature was 80°C programmed to 260°C (5°C/min) then to 300°C .et al. http://chemdata.nist.gov/mass-spc/amdis/) was used for mass spectral deconvolution, which separated peaks from noise and overlapping peaks. Then, the ELU files were subsequently analyzed with an online peak-filtering algorithm . The identification was performed using spectra of individual components transferred to the NIST mass spectral search programs MS Search 2.0, where they were matched against the NIST MS library; for identification, a match quality of 70% was generally accepted. Peak areas of multiple derivative peaks belonging to one compound were summed and considered as a single compound. Normalization to an internal standard peak area was performed before multivariate statistical analyses.Raw GC-MS data were processed as described by Styczynski p<0.05. Partial least-squares discriminant analysis (PLS-DA) was performed in SIMCA-P software  using mean-centered and unit variance-scaled data, which yields a clearer differentiation of each class and enables a less complicated investigation of marker compounds than principal component analysis by rendering the class to each sample group R2), which describes the goodness of fit, and Y predictable variation (Q2), which measures the predictive power of the model.The relative intensities of assigned metabolites by GC-MS analysis were analyzed in each sample. Significant differences in metabolite levels were detected by one-way analysis of variance (ANOVA) using PASW Statistics 18 software  followed by Tukey's significant-difference test. The level of statistical significance was set at http://csbg.cnb.csic.es/mbrole/. The interface used ID conversion utility, which performs the analysis using the metabolites identified by KEGG . We selected Aspergillus niger as a background set. The result contains the list annotation over-represented in the input set with respect to the background set and metabolite-associated p-values.A data functional enrichment analysis with identified metabolic data for biological information was performed using MBRole C. bassiana (20 g) grown to different stages was extracted in screwcap vials with 400 mL of 70% methanol. The samples were irradiated four times in a microwave irradiation machine  for 10 min at 80°C. After extraction, the samples were filtered with filter paper , freeze-dried  for 48 hours and then stored at −80°C for antioxidant activity analysis. The free radical scavenging ability of C. bassiana was determined by following the procedures by Kovatcheva-Apostolova et al. −5 µM 2,2-diphenyl-1-picryl hydrazyl (DPPH)  solution. The mixture was incubated for 30 min in the dark at room temperature. The antioxidant activity of the plant was measured at 515 nm with a microplate spectrophotometer . Free radical scavenging activity (%) was calculated by the following formula:The freeze-dried control is the absorbance without sample, and Asample is the absorbance with sample.An-hexane, respectively. Mass spectral data , amino acids ,organic acids , phosphoric acids (adenosine-5-monophosphoric acid and glycerophosphoric acid), purines , sugars , and others .Samples of fruit bodies at four developmental stages were extracted using 70% methanol and 100% ral data  were ideC. bassiana fruiting bodies in previous studies by NMR n-hexane extracts.As listed in R2Y, while the predictive ability of the model was indicated by Q2Y,respectively R2Y intercept values of 0.242 and 0.164, and Q2Y intercept values of −0.394, and −0.444, respectively. In general, models with an 2YR intercept of less than 0.4 and a Q2Y intercept of less than 0.05 are considered to be valid PLS-DA was performed to compare the metabolic profiles of fruiting bodies at different developmental stages. The PLS-DA model was cross-validated. The goodness of fit and predictive ability of the PLS-DA model were quantified using R2 = 0.96, Q2 = 0.89) in n-hexane extracts . Samples from stage 4 were clearly separated from the other sample groups, mainly by PLS component 1, which indicates that profiles of non-polar metabolites in stage 4 samples were clearly distinguished from the samples at other stages.The metabolic profile of the 70% methanol extracts of fruiting bodies at each developmental stage is represented in the PLS-DA-derived score plot as a single point : threonine, putrescine, serine, valine, fructose, arabinose, ribose, glucitol, isoleucine, lysine, glycine, ornithine, maltose, histidine, glutamine, glucose, aspartic acid, mannitol, glycerophosphoric acid, and proline. The following 13 were associated with ATP-binding cassette (ABC) transporters (p = 0.0000): tryptophan, proline, asparagine, tyrosine, aspartic acid, valine, isoleucine, serine, threonine, lysine, glutamine, histidine, and glycine. The role of aminoacyl-tRNA is to deliver amino acids to the ribosome for the biosynthesis of various proteins. Metabolites associated with purine metabolism , the TCA cycle  and biosynthesis and unsaturated fatty acids and fatty acid  were also described. This suggests that various metabolic pathways are associated with the different developmental stages of cultivated C. bassiana.The most relevant metabolic pathways involved in the metabolite network during the development of analysis .Many metanalysis . As listSaccharomyces cerevisiae and Schizosaccharomyces pombeB. bassiana is reduced when ABC-transporter genes such as Pdr, Mdr, and Mrp are disrupted by mutation The results of the ANOVA conducted to compare differences in the relative levels of the metabolites at various developmental stages are presented in C. bassiana fruiting bodies at different development stages. Further studies on the roles of various transporters at different developmental stages of C. bassiana are needed.Energy-consuming ABC transporters exist in microbial cell membranes. These transporters play important roles in regulating nutrient uptake and secreting toxins or antimicrobial agents C.bassiana fruiting bodies.Higher levels of amino acids associated with purine metabolism, such as glutamine and glycine, were observed at stage 3. Not only are the levels of amino acids such as tyrosine, tryptophan, asparagine and GABA increased at stage 3, but so also are those of purines such as adenosine, inosine, guanosine, and uric acid. Therefore, amino acid metabolism and purine metabolism may play important roles in regulating the development of cultivated The levels of sugars such as fructose, galactose, glucose, and mannose were significantly increased at stage 3 . A carboNectria haematococcaC. bassiana at stage 4 may be attributable to the enhanced usage of the fatty acids as energy resources for ascospores release from the perithecia.As indicated in Among the various fatty acids, the levels of palmitic and linoleic acids which are known key intermediates involved in membrane biogenesis were higher level at stage 3 than at the other stages. It appears that palmitic and linoleic acids accumulate during stage 3 in preparation for ascospore formation.3), were observed at stage 4. Putrescine is a breakdown product of amino acids Higher levels of putrescine and nicotinic acid,  than in samples from other stages B. bassiana prevent an increase in reactive oxygen species and the subsequent damage that they can cause r2 = 0.99) were found in the present study between the content of purine nucleoside compounds , the relative levels of metabolites associated with ABC transporters, aminoacyl-tRNA biosynthesis, and purine metabolism were significantly increased, whereas levels of metabolites associated with lipid metabolism decreased in the ascospores released stage (stage 4).The free radical scavenging activity, which was significantly higher in stage 3 than in the other stages, was positively correlated with purine derivatives such as adenosine, guanosine, inosine and uric acid. Thus, C. bassiana of stage 3 was suggested to be a better resource with higher level of free-radical scavenging activities compared to the other fruiting body samples of C. bassiana at other stages. We suggest that the metabolic profiles during fruiting body development determined in this study provide useful criteria for selecting appropriate harvest points of fruiting bodies of C. bassiana. To our knowledge, this is the first study to investigate the correlation between metabolic profiles and free radical scavenging activities of C. bassiana fruiting bodies according to developmental stage.In this study, we performed metabolic profiling of Figure S1Representative GC-MS spectra of 70% methanol (A), and 100% n-hexane (B) extracts of cultivated C. bassiana at various development stages.(TIF)Click here for additional data file.Table S1Metabolites identified by GC-MS analysis of 70% methanol extracts in C. bassiana fruiting bodies. The relative levels of each metabolite were obtained by dividing the area % of metabolite by the area % of internal standard. Different letters in the same row represent a significant difference. Data are mean ± STD values for triplicate measurements. ND, not detected in the sample.(DOCX)Click here for additional data file.Table S2Metabolite identified by GC-MS analysis of 100% n-hexane extracts of C. bassiana fruiting bodies. The relative levels of each metabolite were obtained by dividing the area % of metabolite by the area % of internal standard. Different letters in the same row represent a significant difference. Data are mean ± STD values for triplicate measurements. ND, not detected in the sample.(DOCX)Click here for additional data file.Table S3Additional list of KEGG pathways from enrichment analysis of metabolite roles.(DOCX)Click here for additional data file.Table S4Quantification of purine contents according to developmental stage of C. bassiana. Data are mean ± STD values for duplicate measurements. Different letters in the same row represent a significant difference.(DOCX)Click here for additional data file.


Lawsonia intracellularis was assessed by investigating the seroprevalence of this pathogen among wild boars in the Republic of Korea. The extent of exposure to L. intracellularis among wild boars (Sus scrofa coreanus) was monitored by a country-wide serological survey using an immunoperoxidase monolayer assay.The importance of the wild boar as a reservoir of L. intracellularis were observed in 165 of 716 clinically healthy wild boars tested. The overall apparent prevalence calculated directly from the sample and the true prevalence calculated based on the accuracy of the test method were 23.0%  and 25.6% , respectively. Serologically positive animals were found in all the tested provinces.In this study, antibodies to L. intracellularis is present in the wild boar population worldwide, even in Far East Asia. Despite the high seroprevalence shown in wild boars, further studies are warranted to evaluate their potential as a reservoir species because seroprevalence does not prove ongoing infection nor shedding of the bacteria in amounts sufficient to infect other animals. It should also be determined whether the wild boar, like the domestic pig, is a natural host of L. intracellularis.Our results confirm that  Lawsonia intracellularis causes porcine proliferative enteropathy (PPE) in domestic pigs (Sus scrofa domestica), and today, PPE is present in all swine-producing areas worldwide 2p(1-p), where Se and Sp are the sensitivity and specificity of the test, respectively, and p is the assumed prevalence. Although we tried to estimate the assumed prevalence from published data on L. intracellularis positivity in the wild boar population, the range of prevalence previously reported was wide, from 59.2 to 9.1% [p = 0.50 to maximize the sample size. A minimal sample size of 208 at the 95% confidence level was required according to the above formula.Wild boar serum sample collection took place between December 2010 and April 2011 by local governments. Samples were selected from national serum specimen resources originally obtained for the purpose of foot-and-mouth disease surveillance as an emergency response during the 2010/2011 foot-and-mouth disease epidemic ,22. Orig to 9.1% ,14. BecaL. intracellularis by IPMA. Using the pathogenic isolate PHE/KK421 (Korean Collection for Type Cultures 10686BP) [L. intracellularis culture plate was incubated with sera diluted 1:30 in phosphate-buffered saline (PBS) for 30 min at 37°C and washed 5 times with PBS, pH 7.2. Peroxidase-labeled goat anti-porcine IgG was diluted 1:1000  in 2% bovine serum albumin and 0.08% Tween 80 in PBS and then added at a concentration of 30 μL/well. The plate was incubated for 45 min at 37°C. The plate was washed again, and a chromogenic  solution was added to each well. The plate was then incubated at room temperature for 20 min. The plate was washed with distilled water three times, allowed to dry, and examined using an inverted light microscope . L. intracellularis-positive and L. intracellularis-negative antiserum controls and a secondary antibody control were included on each plate. Positive samples contained red-labeled bacteria, both in the cytoplasm of infected McCoy cells and in the supernatant. Negative control plates using mock-infected cells were included for each individual serum sample to avoid false-positive results.Blood samples were examined for the presence of antibodies against 10686BP) , IPMA wa10686BP) . BrieflyTrue prevalence (TP) was estimated, as described by Marchevsky et al. -12, usinThe author declares that he has no competing interests.L. intracellularis and McCoy cells and IPMA, wrote the manuscript, conducted analysis of wild boar serum samples and data results, and finalized the manuscript.JYY conceived of the study, designed the experiments, conducted the majority of the experiments in the laboratory including the cultivation of


Hearing loss is one of the most common problems in elderly people. Functional side effects of hearing loss are various. Due to the fact that hearing loss is the common impairment in elderly people; the importance of its possible effects on auditory memory is undeniable. This study aims to focus on the hearing loss effects on auditory memory.Dichotic Auditory Memory Test (DVMT) was performed on 47 elderly people, aged 60 to 80; that were divided in two groups, the first group consisted of elderly people with hearing range of 24 normal and the second one consisted of 23 elderly people with bilateral symmetrical ranged from mild to moderate Sensorineural hearing loss in the high frequency due to aging in both genders.Significant difference was observed in DVMT between elderly people with normal hearing and those with hearing loss (P<0.22). According to the correlation test between Pure Tone Average (PTA) and the mean score of DVMT, increasing PTA resulted in reduction of DVMT score and this result was seen in both genders and all of the studied subjects.Apart from aging, age-related hearing loss has shown significant effect on auditory verbal memory. This result depicts the importance of auditory intervention to make better communicational skills and therefore auditory memory in this population. Hearing loss is one of the most common problems in elderly people. It is the third chronic disease in elderly people after arthritis and hypertension. Based on the United States statistics, up to 40 to 45 % of elderly people older than 60 years old experience some degrees of hearing loss, this range increases to 83%for more than 70 years old people ,2, and iThe dichotic Verbal Memory Test (DVMT) is one of the most valid tests for detecting auditory-verbal memory deficits and differences in memory function between the hemispheres . The higWith regard to the growing geriatric population, high prevalence of hearing loss among this group and the intensive functional effects of hearing loss, it is critical to gain knowledge about the relationship between hearing loss and cognition, in particular, auditory verbal memory to manage old patients with hearing deficit. The knowledge and information about the relation between the hearing loss and memory could be developed by fundamental-application studies like this. And such tests could become more empirical in clinical evaluation via these kinds of studies which can lead to precise consultation and referral of patients with hearing and cognitive impairments; therefore, the elderly people and family members can be helped to understand the problem and provide the means to improve the patients’ quality of life and prevent them from social isolation and depression or dementia. Furthermore, this study can provide a normal value of Persian version of DVMT for aged people with normal hearing and mild to moderate hearing loss of elderly people. Also, in view of the fact that the studies about the hearing loss effects on cognition are minor and in Iran , this study was conducted to evaluate the effects of hearing loss on auditory memory on the elderlies between 60-80 years old.This cross-sectional study was performed on 47 old people aged 60 to 80 years old since 2012 January till 2012 June. The participants included 24 elderly people with normal peripheral hearing (PTA < 25dBHL) and 23 elderly people with mild to moderate symmetrical high frequency age- related hearing loss (55>PTA>25 dBHL). This study was approved by the ethics committee of Tehran University of Medical Sciences. The participants were sampled non-randomly among available elderly people. At first, we went to Omid Aging Institution in Tehran and found the participants; the entrance criteria include (1): Symmetrical hearing thresholds better than 55 dBHL in three frequencies of 1000-2000 and 4000 Hz (2). Normal word recognition score (WRS>90%) in both ears (3). Absence of any aural disease by otoscopy and tympanometry examinations (4). Normal acoustic reflexes in 500 -1000 and 2000 in both ears (4). Right handedness (5) Persian native speaker (6). Literacy level of 9 years and more (7). To eliminate the confounding effect of neurological abnormalities on the results, elderly people with history of head injury or brain surgery and use of psychological medications and Epilepsy based on the person’s expressions were excluded from study.To insure the cognitive health, Persian version of mini-mental state examination (MMSE) was performed for all participants. MMSE is a brief questionnaire that is used to screen for cognitive impairment. The sensitivity and specificity of Persian version of MMSE for cognitive screening of old people are respectively 90 and 84% (in cut off 21) .Pure-tone and speech audiometry were accomplished in a double-walled, sound-treated audiometric booth, using a clinical audiometer (Intracoustic AC40) with supra-aural headphone (Telephonics TDH-39P). Based on hearing thresholds in frequencies of 1000-2000 and 4000, the participants were divided in two groups, the first group included elderly people with normal hearing and the second group included the elderly people with presbycusis.Persian version of DVMT was performed at the Most Comfortable Level (MCL) of hearing by using the two-channel audiometer with CD player compatibility (Interacoustic AC40) with supra-aural headphone (Telephonics TDH-39P). DVMT includes three different lists that each one includes 10 words. The words of each list were presented to right / left ear while the inverse of the same word synchronously presented to the other ear. At the end of each list, the patient was asked to remind and repeat the word regardless of the order in list. The correct answers were inscribed by the examiner, then the memory score was calculated for each list separately; average of three lists as ear score, average of two ears as total score of memory for each person. Kolmogorov-Smirnov test was used to define normal distribution of data. Independent t-test was conducted to compare the mean of the results, and correlation function was done to determine the relationship between PTA and DVMT. For statistical analysis, software Spss.17 was used based on 0.05 significant level.By using Kolmogorov-Smirnov test, all values of the test data showed normal distribution (P>0.05).In the current research, we studied hearing loss effect on DVMT. The average of DVMT score in aged people with normal hearing was 4.62 ±.75 and in aged people with hearing loss was 4.06 ±.86. Correlation between subjects PTA and DVMT score average is shown in Age-related hearing loss or presbycusis is one of the most common sensory deficits in the elderly, and as an increasingly important public health problem, it results in reduction in memory and quality of life . MoreoveCorrelation between PTA and DVMT showed that increasing PTA resulted in the reduction of test score in all of the studied persons and in both genders separately. In fact more hearing loss causes more reduction in memory function.Our result was consistent with Van Boxtel and colleagues in 2000 which studied the amount of immediate and delayed recall in an auditory verbal learning paradigm, their results indicated lower performance in subjects with mild to moderate hearing loss. And this result was observed even when the investigators make the situations clear in background noise and make improvement in signal and noise ratio . An assoIt is worth noting that the superiority of our study to previous research was audiometry evaluation based on available standard in vocal booth treatment with clinical audiometer. Although most studies utilized portable or screening audiometers or participants tested under varying environmental conditions , the effect of biased or imprecise assessments of hearing thresholds would likely decrease sensitivity to detect associations due to increased variance. The other superiority was utilizing a verbal test which can show the relationship between PTA and auditory memory test. There are different explanations for existence of hearing loss effect on cognitive function: first is the "common cause" hypothesis which is about brain aging and its adverse effect on many areas of the brain. This hypothesis states that auditory and cognitive declines are the characteristic of neural degeneration in many areas. The aging of the central nervous system may have a direct impact on many different brain functions . It shouThe current study depicted another thought provoking result in which the DVMT scores were different between males and females. In many studies, it was observed that females performed better than males in immediate and delayed object recall and visual memory. In contrast males performed better in digit span, abstractive thought and spatial skills . In our Female’s performance in verbal memory test could be due to the difference in brain structures between two groups. So far several studies showed the difference between structural and functional brain of males and females . CompariOur study also had some limitations as follows; The results were based on cross-sectional data instead of longitudinal study and variety of designs in cross-sectional study like using population- or selected samples, were not substituted for high-quality longitudinal data, and longitudinal study must be used to explore emergent hypotheses. Another limitation was the range of our subjects with hearing loss since the subjects had mild to moderate hearing loss. To investigate the hearing loss effect it also could be thought appropriate to evaluate old people with poorer hearing. This research was conducted to study the relationship between sensory loss (i.e. hearing) and cognitive function (i.e. auditory memory). To investigate the hearing loss effect on auditory verbal memory, a kind of verbal memory test known as DVMT was used. The result showed that there were s significant relationship between hearing loss and auditory memory performance. Based on these results, it is suggested that the auditory-verbal memory assessments can be used as a supplementary tool for better management in clinical evaluation of geriatric patients; furthermore referral for auditory assessments is suggested in patients with cognitive impairment in psychological clinics.


Newborn hearing screening leads to the early detection of hearing impairment. The aim of screening is to decrease or remove the effect of hearing impairment on development of speech and language by timely diagnosis and effective treatment. A number of risk factors lead to delayed start of decreased hearing ability including: 1. Congenital infection with cytomegalovirus (CMV) virus, 2. Meningitis, 3. Mumps, 4. Positive family history, 5. Head trauma, 6. Chemotherapy,7. Syndrome pertaining to delayed start of decreased hearing. Unfortunately, lack of attention to early diagnosis of hearing impairment is becoming a general health problem. No research has yet been carried out relating to the knowledge of pediatricians on this issue, particularly the importance of hearing impairment and hearing screening. The aim of this study was to determine the attitude to newborn hearing screening among pediatricians.This cross-sectional, descriptive-analytic study was conducted in Isfahan in 2012 among 300 pediatricians and final-year pediatric residents. An adjusted 22-question version of the Early Hearing Detection and Intervention (EHDI) questionnaire was used to collect data. The validity and reliability of the EHDI questionnaire was previously demonstrated by Boys Town National Research Hospital and its Farsi translated version was validated by the EDC Center at the Isfahan University of Medical Sciences.In our study, 83% of pediatricians agreed on the importance of hearing impairment screening for all infants. However 65% were not aware of special needs for hearing-impaired patients.Newborn hearing impairment and deafness screening is important, irrespective of the costs, and lack of timely diagnosis results in both individual and social consequences. The majority of physicians use textbooks to gain information about hearing screening, but recognize that this is insufficient. Although it is now one of the most useful tools for gathering and applying new information, the physicians in our study rely very little on the Internet as a source of information. Hearing impairment, a reduction in hearing ability, tends to be an invisible and hidden human disability, but its consequences can be very severe ,2. It isLack of timely diagnosis of hearing impairment among infants can lead to considerable individual and social complications including ,8:1. Delay in natural development of speech and language and speech disorders.2. Learning disorders, academic failure, reading and writing problems.3.Communicational and emotional-behavioral disorders, delay in social-cultural growth.4. Unsuitable communication with parents and friends, misunderstanding and bad behavior towards these people.A Joint Committee of the American Academy of Pediatricians has recommended that all infants be screened by hearing tests to diagnose of hearing impairment early . Timely As primary care physicians, general practitioners (GPs) are the main route to educate and encourage parents to seek hearing screening and also to follow-up hearing-impaired infants. This is because GPs understand the concept of hearing screening the importance hearing and early detection of hearing disorders, and appropriate routes for patient referral . OlusanyThe principal motivation in newborn hearing screening programs is early detection of hearing impairment and accordingly early intervention for rehabilitation. Yoshinaga-Itano et al. reported that infants diagnosed before 6 months who participated in rehabilitation programs have better acquired skills such as pronunciation, understanding, mathematical reading, influential social behaviors, and communication with people other than their families ,14.Lack of hearing impairment diagnosis and late therapeutic intervention is often accompanied by increasing learning risks, greater treatment costs, and loss of productivity ,11,13.Since pediatricians are at the frontline in dealing with hearing-impaired children and their families, the current research aims to study the knowledge of specialists and final-year residents in terms of the importance of hearing impairment and deafness screening.This cross-sectional, descriptive-analytic study was conducted in 2012 in Isfahan. The target population was all pediatricians and final-year pediatric residents responsible for the care of infants. According to statistical reports of the Isfahan Vice Chancellor for education and medical treatment, approximately 290 qualified pediatricians and 10 senior assistants in pediatrics were working in this city at the time the study was conducted.Therefore, the inclusion criteria were pediatricians and final-year pediatrics assistants practicing in infant care, who declared their consent to participate in the study. An adjusted 22-question version of Early Hearing Detection and Intervention (EHDI) questionnaire was used to collect data. Validity and reliability of EHDI questionnaire have previously been demonstrated by Boys Town National Research Hospital and its Farsi translated version was validated by the EDC Center at the Isfahan University of Medical Sciences .Questionnaires in addition to required descriptions were given to pediatricians at their workplace, and completed forms were returned during a subsequent visit. Collected data were analyzed by SPSS20. As there was no drug intervention in this study, this research is consistent with medical ethical issues. Close to twenty-five pediatricians did not participate in this study because of a lack of time In response to the question, “What percentage of your practice is accounted for by infants?”, 17 (5.7%), 74 (24.6%), 92 (30.7%), and 117 (39%) pediatricians responded >75%, 50–75%, 25–50%, and <25% of respectively. In response to the question, “How important is screening of infants for permanent hearing impairment”, 25 (83%) considered it important, 38 (12.7%) considered it to be of little importance, and 11 individuals (3.6%) responded,“Unimportant”.The mean age of the respondents was 48.2±7.4 years, and the population consisted of 257 (85.7%) males and 43 (14.3%) females. Demographic data for the study population are presented in .Results showed that 210 (70%) pediatric- cians had seen between two and five infants with permanent hearing impairment and 90 (30%) had seen fewer that two infants within the previous three years. Further, 291 (98%), 280 (92%), and 5 (2%) doctors had referred infants with permanent hearing impairment to an ear, nose and throat (ENT) specialist, audiologist, and neurologist, respectively.A total of 285 pediatricians (88%) received more than 90% of referred infants hearing screening results while 15 pediatricians (12%) received no results.Responses to the question “What are your primary references for hearing screening?” are shown in Hearing impairment is one of the most prevalent congenital disorders and, if it is not detected, it can have an adverse effect on speech, language, and cognition. According to studies in Iranian infants in Tehran published in 2004, it was estimated that 1–4 infants out of 1,000 births in a normal care unit and 2.5–4.5 infants out of 100 births in an intensive care unit have two-sided severe sensorineural hearing impairment . Also a In the study by Nicole et al. investigating physicians in Minnesota in 2006, 80% considered the hearing screening of all infants important and 75% considered screening valuable, regardless of cost. However in our study, 83.7% of pediatricians recognized that hearing screening of all infants is important and 96.4% of them responded yes to the question “Is hearing screening valuable with any expenditure?”. Furthermore, 55% of Minnesotan physicians were sure about their description of the results of screening and sufficient knowledge for dealing with parents. In our study 48.3% were very sure about screening, 43.3% considered they had a lot of knowledge and 8.4% had a moderate knowledge of screening. Our results also showed that the most prevalent risk factors for delayed hearing impairment among infants are meningitis (98.3%), family history of hearing impairment (92.7%), history of cytomegalovirus (CMV) infection (85%), congenital syphilis (67%), hospitalization in a neonatal intensive care unit (NICU) for more than 48 hours (53.7%), hypotonia (29%), cleft palate (26.7%), chronic heart disease (23.3%), repeated exposure to cold (23%), and mother's age >48 years old (17.7%). In other studies, the first risk factor was meningitis and then family history of hearing impairment and CMV history. In a study of American physicians reported in 2006 by Moeller et al., 99% of respondents said that the most prevalent risk factors were meningitis, family history of hearing impairment, and CMV history. Moellor also reported that 75.8% of physicians referred permanent hearing-impaired infants to an ENT specialist, 41.3% to an audiologist, 8.9% to a geneticist, and 7.1% to a neurologist. However in our study, 98% of physicians referred patients to an ENT specialist, 92% to an audiologist and 2% to a neurologist. Accordingly, we can conclude that our physicians did not refer infants with permanent hearing impairment to a geneticist or do not have sufficient knowledge of this option.Hearing screening should ideally be performed before the infant reaches the age of 1 month. Detection and use of hearing aid equipment should be carried out before 3 months of age, and therapeutic intervention before the age of 6 months ,18. ThisNewborn hearing screening programs help to determine the level of decreased hearing ability and allow physicians to take the appropriate action in cooperation with an ENT specialist, pediatrician, neurologist, geneticist, and audiologist. According to the results of this study, physicians understood the importance of infant hearing screening and considered it valuable irrespective of its cost; they were also aware of its role in the prevention of individual and social complications. Therefore, suitable frame- works need to be established to allow infant hearing screening to become a financial possibility. According to the results presented herein, the majority of physicians used textbooks to access information relating to hearing screening, but they recognized that this was an insufficient resource. Although it is now one of the most useful tools for gathering and applying new information, the physicians in our study rely very little on the Internet as a source of information. Furthermore, the physicians in our study did not have sufficient knowledge about the special needs of children with hearing impairment, and believed that they could increase their knowledge by using additional sources of reference.