The plasmid was used for transfection of HCT116 colorectal carcinoma cells, while data from non-transfected and pEGFP-N1-transfected cells is also shown. Immunoblotting data of lysates from non-transfected controls and HCT116 cells transfected with pEGFP-N1 and phCL-EGFP, showed stable expression of cathepsin L-enhanced green fluorescent protein chimeras, while endogenous cathepsin L protein amounts exceed those of hCL-EGFP chimeras. An effect of phCL-EGFP expression on proliferation and metabolic states of HCT116 cells at 24 h post-transfection was observed.The endo-lysosomal cysteine cathepsin L has recently been shown to have moonlighting activities in that its unexpected nuclear localization in colorectal carcinoma cells is involved in cell cycle progression   Specifications TableValue of the data•This article provides information on the construction of a vector coding for human cathepsin L-EGFP chimeras, and it describes the insert׳s sequence.•The data provide detailed information on the molecular forms of cathepsin L chimeras with enhanced green fluorescent protein that are expressed in the human colorectal carcinoma HCT116 cell line.•The article provides data on the effects of cathepsin L-EGFP expression in HCT116 cells with regard to proliferation rates and metabolic activity.DataEGFP-tagging is used to visualize transport and trafficking of cellular proteins including cysteine cathepsin proteases We have shown that cathepsin L is sorted and mis-trafficked to the nucleus of HCT116 cells as full-length protein and in proteolytically active form 1pEGFP-N1 by NheI and BamHI restriction. Here, we share the relevant sequence data of phCL-EGFP. The vector is introduced through lipofection to express chimeric, EGFP-tagged cathepsin L in HCT116 cells. This colorectal carcinoma cell line was further analyzed with regard to proliferation and metabolic activity by MTT-assays upon transfection with phCL-EGFP in comparison to non-transfected and pEGFP-N1-transfected cells. The molecular forms of protein chimeras expressed in HCT116 cells were identified by immunoblotting of whole cell lysates separated by SDS-PAGE.In this article, we describe the construction of a vector encoding human cathepsin L-EGFP (hCL-EGFP) with cDNA derived from cathepsin L mRNA of human HaCaT keratinocytes upon insertion into the multiple cloning site of 1.1The human keratinocyte line HaCaT  was provided by Prof. Dr. Petra Boukamp  and cultured in Dulbecco׳s modified Eagle׳s Medium  supplemented with 10% fetal calf serum . Total RNA was isolated from phosphate-buffered saline (PBS)-washed confluent HaCaT cell cultures using TRIZOL reagent  with subsequent chloroform and isopropanol purification. The RNA pellet was washed with 75% ethanol and finally re-suspended in 30 µl of DEPC-treated water . The RNA concentration was determined by nanodrop microphotometry  before aliquots were either directly used or snap-frozen in liquid nitrogen and stored at −80 °C.1.2NheI” (5′-aca cag gtt tta aaa cat gaa tcc tac a-3′) and “CL rev BamHI” (5′-agc tac ccc act gtg tga gct ggt gga-3′), 0.125 mM each of dNTPs, and 0.4 U Phusion DNA polymerase . By this cloning strategy, restriction sites for NheI and BamHI were introduced and the stop codon was removed, thus eventually yielding the sequence for a chimeric protein with the full-length cathepsin L sequence covalently connected to the EGFP tag by a spacer peptide linker. The final PCR product and pEGFP-N1 plasmid were each digested with NheI and BamHI . The restricted cDNA was ligated into the linearized vector using T4-DNA ligase (MBI) in the presence of ATP-containing reaction buffer (MBI) at 4 °C overnight, followed by ligase inactivation at 65 °C for 10 min. The ligated plasmids were transformed into competent E. coli JM 109, and kanamycin-resistant clones were used for isolation of vector cDNA , which was eluted and stored in 10 mM Tris–HCl at pH 8.0. Control digests of the isolated plasmids were performed with NheI and BamHI, each used at 0.25 U/µg plasmid DNA, for insert restriction, and additionally EcoRI with a restriction within the insert was used  before analyzing on 1% agarose gels by electrophoresis. Finally, sequencing using the primers “CMV-Profor” (5′-aaa tgg gcg gta ggc gtg-3′) and “EGFP-Nrev” (5′-cgt cgc cgt cca gct c-3′) confirmed the correct product .Total RNA at a concentration of 0.05 µg/µL was reverse transcribed with a cDNA cycle kit  using oligo dT primers and AMV reverse transcriptase. Amplification of the complete cathepsin L coding sequence was by PCR with 500 ng cDNA using 0.5 µM each of the primers “CL for 1.32-atmosphere  in RPMI-1640 medium (Biowhittaker™) supplemented with 10% fetal calf serum . Transfection with pEGFP-N1 and phCL-EGFP was performed using X-treme GENE HP DNA transfection reagent  following the manufacturer׳s protocol.The human colorectal carcinoma HCT116 cell line was purchased from ATCC . The cells were cultured at 37 °C in a 5.0% CO1.4Cell proliferation rates and metabolic activity levels of transfected HCT116 cells were examined by a colorimetric assay in which the yellow 3--2,5-diphenyltetrazolium bromide (MTT) is reduced to form purple formazan crystals by intracellular NAD(P)H-oxidoreductase. The assays were performed in duplicates and repeated at least twice as previously described 1.5Transfected HCT116 cells were used 24 h post-transfection, and whole cell lysates were prepared in PBS containing 0.2% Triton X-100 as described before 2phCL-EGFP coding for EGFP-tagged cathepsin L was expressed in HCT116 cells and sorted to the nucleus [Cathepsin L has been shown to be mis-trafficked in HCT116 cells and to enter the nucleus as an unexpected scene of potential action 2.1pEGFP-N1 vector , resulting in the fusion of sequences coding for cathepsin L and eGFP in phCL-EGFP. The cloning strategy  and upstream (starting 3′) in the vector  revealed 100% identity, confirming that cathepsin L-coding cDNA was inserted into the pEGFP-N1 vector as shown in the vector map of phCL-EGFP  and heavy chain (HC) of the two-chain form of the protease. Lysates of phCL-EGFP-transfected HCT116 cells featured an additional band at 65 kDa that was recognized by cathepsin L-specific antibodies (pEGFP-N1. The data indicates that the band at 65 kDa is representative of the hCL-EGFP chimeric protein which was stable for 24 h in HCT116 cells because no degradation bands could be observed in immunoblots with cathepsin L-specific antibodies (To identify the molecular forms of the cathepsin L-EGFP chimeras translated in HCT116 cells, immunoblotting was performed with whole cell Iysates of non-transfected cultures and with those expressing tibodies , and whitibodies .


Luteinizing hormone-releasing hormone (LHRH) neurons and fibers are located in the anteroventral hypothalamus, specifically in the preoptic medial area and the organum vasculosum of the lamina terminalis. Most luteinizing hormone-releasing hormone neurons project to the median eminence where they are secreted in the pituitary portal system in order to control the release of gonadotropin. The aim of this study is to provide, using immunohistochemistry and female brain rats, a new description of the luteinizing hormone-releasing hormone fibers and neuron localization in the anterior hypothalamus. The greatest amount of the LHRH immunoreactive material was found in the organum vasculosum of the lamina terminalis that is located around the anterior region of the third ventricle. The intensity of the reaction of LHRH immunoreactive material decreases from cephalic to caudal localization; therefore, the greatest immunoreaction is in the organum vasculosum of the lamina terminalis, followed by the dorsomedial preoptic area, the ventromedial preoptic area, and finally the ventrolateral medial preoptic area, and in fibers surrounding the suprachiasmatic nucleus and subependymal layer on the floor of the third ventricle where the least amount immunoreactive material is found. The luteinizing hormone-releasing hormone (LHRH) is a gonadotropin releasing hormone (GnRH), which acts on the pituitary hormones as a follicle stimulating hormone (FSH) and luteinizing hormone (LH), which act on the gonads, . The GnRThe preoptic area (PA) is part of the anterior hypothalamus and is confined to the anteroventral region of the third ventricle (AV3V); the PA is divided into, the medial preoptic area (MPA) and lateral preoptic area (LPA). The MPA makes its morphological appearance at eight weeks of gestation in humans and is located in the periventricular regions of the anterior hypothalamus covering the organum vasculosum of the lamina terminalis (OVLT) , 10. TheThe OVLT , 19 beloBrains from five female Wistar rats from Charles River Laboratories España S.A.  of 6 months of age were used. Rats were kept under lighting conditions of 12:12, and food and water were provided ad libitum. Rats were sacrificed at diestrus stage , and bef The sections of five coronal cephalocaudal anatomical levels of the anterior hypothalamus  were simThe immunohistochemistry slides were converted to digital images by using an LEICA DMRB photomicroscope with an LEICA DC 300 F camera (Germany). Image analysis was completed by ImageJ . The “mean gray value” was measured from the selected areas for all stained tissue. This value gives the average stain intensity in grayscale units for all threshold pixels. The immunohistochemistry statistical study was conducted using the IBM SPSS statistic 19 software (one-way ANOVA).Many LHRH fibers and neurons were found in different parts of the preoptic hypothalamus; the neurons presented a monopolar or bipolar morphology; the dendrites were uncomplicated, without branches and roofed with spines Figures . The distribution of LHRH cells and fibers is described with the use of the Paxinos and Watson atlas of the rat , the Paxa  till the level d, where the smallest amount of immunoreactive material was found  was found in the OVLT at the level as found .  The findings here agree with morphological studies which report that the GnRH neurons have long and simple uni- or bipolar dendrites typically covered in spines , 32. The In view of the prevailing significance of the GnRH neurons, it is necessary to know their anatomical localization to fully understand their structure and function in general, specifically in the sexual dimorphism. At the same time, it is unclear whether the medial preoptic area is a diencephalic or a telencephalic structure because the MPA is located in the anterobasal forebrain or prosencephalon during the early stages of its development. But in the following stages, when the diencephalon and telencephalon develop and differentiate from the prosencephalon, the preoptic area is anatomically localized in the anterior hypothalamus , 11, altc and d , and therefore, this is the terminology used in this study. Positive LHRH cells and fibers located in different parts of MPA and in OVLT such as dorsomedial preoptic area (DMPA), Figures  Figures , would cIn view of that described above, one could conclude that the exact denomination of the LHRH localization is peculiar; however, one could say that the greatest amount of LHRH immunoreactive material (LHRH-ir) was found in the OVLT and decreased cephalocaudally in the preoptic hypothalamus. LHRH immunoreactive material was found in the following parts: dorsomedial preoptic area, ventromedial preoptic area, and ventrolateral medial preoptic area, and in fibers surrounding the suprachiasmatic nucleus and subependymal layer on the floor of the third ventricle where the least amount of LHRH fibers and neurons was found.


Neoseiulus barkeri, exhibited feeding behavior on the entomopathogenic fungus Beauveria bassiana conidia through DNA extracts. Using transmission electron microscopy, we determined that the majority of conidia found in the mite gut tended to dissolve within 24 h post ingestion, suggesting that the conidia had probably lost their viability. To our knowledge this is the first report of feeding behavior of phytoseiid mites on entomopathogenic fungus. The findings expand our knowledge of fungus–predator interactions.Interactions between fungal entomopathogens and pest predators are particularly relevant in control of agricultural insect pests. In a laboratory study, we confirmed that the predatory mite,  The use of multiple natural enemies has been recommended to control insect pests in integrated pest management programs2Beauveria bassiana is known to be effective in controlling many arthropod pestsB. bassiana has shown to have no pathogenicity to the predatory mite Neoseiulus barkeri (Acarina: Phytoseiidae)The entomopathogenic fungus N. barkeriTyphlodromus pyriAmblyseius swirskiiMost previous studies on the interactions between fungal pathogens and predatory mites have focused on the fungal infectivity to predators81012N. barkeri on B. bassiana. In addition, ingested B. bassiana conidia inside the mite gut were morphologically compared with normal to reveal the viability and pathogenicity of conidia as well as transmission efficiency after ingestion.This study seeks to investigate feeding behavior of B. bassiana inoculation, conidia were ingested by N. barkeri through long narrow esophagus for entry into broad midgut under a microscope (see B. bassiana (>95%). Phylogenetic analysis showed that it was closely related to the Bb3113 strain.After cope see . Fungal cope see . A BLASTAs demonstrated by transmission electronic microscopy (TEM), residual conidia were observed in the intestines of treated mites, but not in the untreated mites . FurtherB. bassiana conidia depends on the water activity of the nutrient liquidCompatibility between entomopathogenic fungi and predatory mites is of critical importance for controlling their shared pests. For example, predators may also function as dissemination agents in exposing susceptible hosts to conidia leading to infection in the pest speciesB. bassiana were not able to penetrate the cuticle of N. barkeriB. bassiana has no pathogenicity to N. barkeri through ingestion. The B. bassiana–fed mites became smaller in body size after 24 h and more transparent within 48 h compared to normal mites fed with mite prey. These observations implicate that the conidia ingested by the mites were of poor nutritional value.The pathogenicity of a fungus to a host insect is mainly caused by the conidia penetrating the insect’s cuticleB. bassiana and predatory mites. That is, the feeding behavior of N. barkeri, on B. bassiana conidia would reduce the amount of sprayed conidia should the two control agents be presented simultaneously in an agroecosystem.Predator-fungus interactions are complex. They can interact directly through conidia dissemination with positive consequences in suppressing a pest population. Conversely, they may interact indirectly mediated by their common prey or fungal conidia causing antagonistic effects. The present study provides new examples of negative interactions between No specific permissions were required for these locations/activities.None of the species used in this study are endangered or protected.B. bassiana SZ-26 was derived from Ostrinia nubilalis  larvae collected in Suizhong, Liaoning, China (2010). Fresh cultures were maintained on Sabouraud dextrose agar (SDA) for conidiation at 26 ± 1 °C under continuous darkness. Conidial concentration (1 × 108 conidia mL−1) was prepared with sterile 0.05% Tween-80. Viability was confirmed on SDAFrankliniella occidentalis, but no infectivity to N. barkeriThe isolate of N. barkeri was maintained in the Laboratory of Insect Natural Enemies, Institute of Plant Protection, Chinese Academy of Agricultural Sciences. N. barkeri stock colonies were cultured on excised kidney bean (Phaseolus vulgaris) leaves in a covered plastic container (15 cm × 15 cm × 10 cm)Tyrophagus putrescentiae as prey. The newly emerged adult females were used in the feeding experiments.A colony of B. bassiana suspension was sprayed into each chamber using a 2 ml hand pressure sprayer. Once the conidia were deposited on the leaf, 10 N. barkeri mites were transferred into each feeding chamber and the second piece of plexiglass was placed on top. The layers were tightly clipped together on both ends to avoid mites escaping. The feeding behavior of mites on the B. bassiana suspension was observed under a microscope. To analyze the conidia in the gut of mites, the mites were allowed to feed on the B. bassiana for 30 minutes. They were then removed and the mites were eluted with sterile 0.05% Tween-80 to remove any residual conidia adhering to the surface of the mites. Ten mites were then manually homogenized in liquid nitrogen and the DNA extracted according to the manufacture’s instruction using the Plant Genomic DNA Kit . The internal transcribed spacer (ITS) was amplified and sequenced with universal primers ITS4 (TCCTCCGCTTATTGATATGC) and ITS5 (GGAAGTAAAAGTCGTAACAAGC)B. bassiana suspension and then immediately eluted with sterile 0.05% Tween-80. Fungal DNA was extracted as described above. DNA sequences were assembled and edited using Sequencher 4.1 . The output DNA sequences were BLAST compared using NCBI and phylogenetic analysis was performed by MEGA.Experiments were conducted using two uniform pieces of plexiglass (6 cm × 5 cm × 4 mm). A water-saturated filter paper was placed on one piece of plexiglass, and a freshly excised kidney bean leaf disc was placed topside down on the surface of the filter paper. A 2.5 cm diameter hole was punched in another piece of plexiglass and placed on top of the leaf, forming a feeding and observation chamber between the two pieces of plexiglass. Prior to placing the second piece of plexiglass, a B. bassiana, treated and untreated mites were fixed in 5% glutaraldehyde in a soda-cacodylate buffer at room temperature for 12 h. The mites were then rinsed five times with 70% ethyl alcohol, and dehydrated through an ascending series of ethyl alcohol  before embedding in Spurrs’ resin. Ultrathin sections from the intestine were cut and collected on carbon-stabilized Formvar-coated grids. The samples were then stained negatively with uranyl acetate solution and dried under silica gel. Thin sections were cut from the mite intestine using a microtome. The morphological structure of residual conidia from the gut of treated mites was observed via TEM and compared with non-ingested conidia. All the mite specimens were starved for 24 h prior to the test to minimize the influence of extraneous intestinal material.To confirm the viability of conidia collected from the mite guts after 24 h exposure to How to cite this article: Wu, S. et al. Insight into the feeding behavior of predatory mites on Beauveria bassiana, an arthropod pathogen. Sci. Rep.6, 24062; doi: 10.1038/srep24062 (2016).


GJB2 gene) and 97 subjects with GJB2 genotype wt/wt in the Republic of Sakha/Yakutia . This subarctic territory was found as the region with the most extensive accumulation of mutation IVS1+1G>A in the world as a result of founder effect in the unique Yakut population isolate. The GJB2 gene resequencing and detailed audiological analysis in the frequency range 0.25, 0.5, 1.0, 2.0, 4.0, 8.0 kHz were performed in all examined subjects that allowed to investigate genotype-phenotype correlations between the presence of single mutation IVS1+1G>A and hearing of subjects from examined groups. We revealed the linear correlation between increase of average hearing thresholds at speech frequencies  and age of individuals with GJB2 genotype IVS1+1G>A/wt . Moreover, the average hearing thresholds on high frequency (8.0 kHz) in individuals with genotype IVS1+1G>A/wt (both sexes) were significantly worse than in individuals with genotype wt/wt (p<0.05). Age of hearing loss manifestation in individuals with genotype IVS1+1G>A/wt was estimated to be ∼40 years . These findings demonstrate that the single IVS1+1G>A mutation (GJB2) is associated with age-related hearing impairment (ARHI) of the IVS1+1G>A carriers in the Yakuts.Age-Related Hearing Impairment (ARHI) is one of the frequent sensory disorders registered in 50% of individuals over 80 years. ARHI is a multifactorial disorder due to environmental and poor-known genetic components. In this study, we present the data on age-related hearing impairment of 48 heterozygous carriers of mutation IVS1+1G>A ( Age-related hearing impairment (ARHI), or presbyacusis, is a common sensory disorder characterized by bilateral sensorineural hearing loss more evident at high frequencies GJB2 gene coding protein connexin 26 (Cx26) in homozygous and compound-heterozygous states are responsible for a significant part of inherited autosomal recessive forms of hearing loss in different populations GJB2 gene (in heterozygous state) have influence on hearing function GJB2 gene – c.35delG, p.Met34Thr, c.167delT (most frequently registered in Europe), and c.235delC single mutation (the study was carried out in China) – had controversial results Genes already known to be associated with hearing impairment are not investigated enough for the presence of genetic associations with ARHI. Mutations in GJB2 gene is relatively rare, and audiological analysis of hearing threshold in carriers of this single mutation has not been performed previously. High rates of IVS1+1G>A mutation (2–11.7%) were found among some indigenous populations of Eastern Siberia, Russia , with the highest carrier frequency (11.7%) in isolated Yakut population GJB2 gene mutation.Splice site mutation IVS1+1G>A in GJB2 mutation IVS1+1G>A carriers versus individuals with normal GJB2 genotype in Yakut population.The purpose of this study is to analyze age-related hearing alteration in GJB2 gene mutations ; had no recent history of fever, otitis media, tinnitus; and had no obvious contacts with occupational noise. From those 145 individuals based on genotyping data we formed two groups: Cx26-H – the group included the carriers of single mutation IVS1+1G>A ; and Cx26-Wt – the group of non-carriers of the IVS1+1G>A mutation . Subsequently, we stratified these two groups according to age and sex for the purpose of correct comparison  and informed the potential participants about the aims of upcoming study. We also invited the parents and siblings of deaf children homozygous for the IVS1+1G>A mutation, as they could be the carriers of this mutation. All the volunteers  passed otologic-audiological evaluation and genotyping for the mparison .0.5,1.0,2.0,4.0 kHz separately.Audiological examination was conducted on all 214 individuals (n = 428 ears). Otologic examination was performed with the «Basic-Diagnostik-Set» , tuning fork standard set. Acoustic impedance was measured with impedance meter . Air conduction thresholds were measured on frequencies 0.25, 0.5, 1.0, 2.0, 4.0, 8.0 kHz and bone conduction thresholds – on frequencies 0.25, 0.5, 1.0, 2.0, 4.0 kHz with audiometer «MAICO ST20»  in the same conditions for all participants. The results were assessed separately for each ear on air conduction thresholds as on all measured frequencies, and speech range of frequencies – PTAGJB2 gene mutation analysis. Amplification of the coding (exon 2) and noncoding (exon 1) and flanking intronic regions of GJB2 gene was conducted with PCR on thermocycler «MJ Mini» (Bio-Rad) using appropriate primers GJB2 gene are shown in the A total of 214 genomic DNA samples, which were extracted from leukocytes of peripheral blood, were used for GJB2 gene were summarized in the unified database. Regression and correlation analysis of «age-hearing threshold» association were assessed with Spearman's rank correlation coefficient sr. Significance of hearing thresholds on measured frequencies was statistically evaluated with the Mann-Whitney U test using software STATISTICA version 8.0.  and Biostatd . Differences were statistically significant when p<0.05.The results of audiological examination and mutation analysis of Written informed consent was obtained from all individuals. This study was approved by the local Committee on Biomedical Ethics of Yakut Scientific Center of Complex Medical Problems, Siberian Branch of the Russian Academy of Medical Sciences, Yakutsk, Russia .0.5,1.0,2.0,4.0 kHz» in IVS1+1G>A single mutation carriers (group Cx26-H) and non-carriers (group Cx26-Wt), for females and males separately. Statistically significant (p<0.05) correlation of increase in hearing threshold according to age was detected in IVS1+1G>A carriers  and not observed in non-carriers    A, B.0.5,1.0,2.0,4.0 kHz» was performed in these age cohorts (p<0.05) correlation was observed in hearing thresholds of individuals from group Cx26-H at ages 40–49 years , and 50–59 years   . Thus, h0.5,1.0,2.0,4.0 kHz) did not identify differences between groups Cx26-Wt and Cx26-H at age 20–39 years . However, statistically significant differences in hearing thresholds were observed at age 40–59 years in both groups Cx26-Wt and Cx26-H   .Following analysis at all measured frequencies  revealed that in group Cx26-Wt averaged audiological profile of females and males aged 20–39 and 40–59 years had a shape of flat curve. Averaged audiological profile of females and males aged 20–39 years in group Cx26-H had a shape of sloping curve, aged 40–59 years – steep-sloping curve in high-frequent range (4.0–8.0 kHz) .p<0.05) differences were observed at frequency 8.0 kHz in both sexes and at frequency 4.0 kHz – in females  . However females .p<0.05) in hearing thresholds at frequencies 4.0–8.0 kHz between groups Cx26-Wt and Cx26-H were observed in females and males aged 40–59 years . Moreover, median value of hearing threshold was ≥20.0 dB in single mutation IVS1+1G>A carriers at 4.0–8.0 kHz for females, and at 8.0 kHz – for males  and individuals with normal GJB2 genotype (n = 29) in Italy, however, this study failed to find a correlation of hearing threshold increase with age GJB2 c.35delG heterozygous females in comparison with controls GJB2 single mutations, c.35delG and p.Met34Thr (c.101T>C), conducted in a UK population from 1991 to 2011 with three-step audiological test of children aged 3, 7 and 11 years (9202 children) showed that hearing thresholds on extra-high frequency (16.0 kHz) in c.35delG and p.Met34Thr (c.101T>C) carriers was significantly worse than in children without GJB2 gene mutation and tend to increase with age Moreover, there is a study that detected statistically significant differences in mean hearing thresholds on high frequencies (6.0 and 8.0 kHz) between mutation c.35delG carriers (n = 31) and individuals with normal A recently published study provides the data comparable to our results. The study carried out in China (2011) showed that in c.235delC heterozygous carriers hearing for high-frequency (4.0 and 8.0 kHz) increases at the age of 30–59 years and hearing thresholds in the intermediate frequencies may deteriorate over the age of 40 0.5,1.0,2.0,4.0 kHz) linearly increase (p<0.05) with age in the heterozygous carriers of single site splicing mutation IVS1+1G>A (GJB2 gene) of both sexes. This correlation is more expressed in males, which probably associated with physiological features and environmental factors. Moreover, audiometric analysis showed that hearing thresholds at high frequency (8.0 kHz) in heterozygous for IVS1+1G>A females and males aged 20–39 years were definitely worse (p<0.05) than in non-carriers. We also found that hearing on lower frequencies (1.0–2.0 kHz) was affected in both heterozygous for IVS1+1G>A females and males aged 40–59 years . Thus, we identified that hearing acuity of single mutation IVS1+1G>A carriers on particular frequencies was decreased compared to non-carriers, regardless of sex and age. Splice site mutation IVS1+1G>A, probably leads to a deficiency of normal Cx26 molecules that form intercellular gap junction channels, and this results in impaired recycling of potassium ions in the cochlea with a local organ of Corti intoxication Our study provides the first evidence that in a relatively isolated Yakut population the average hearing thresholds at the speech frequencies (PTA59 years . At the GJB2) is associated with age-related hearing impairment (ARHI) of the IVS1+1G>A carriers among the Yakuts.Despite the controversial results of previous audiological analysis of the c.35delG mutation in heterozygous carriers from Caucasian populations


We used hearing tests and peripheral blood sample analyses to characterize the pathology of idiopathic sudden sensorineural hearing loss (ISSNHL) and to identify possible prognostic factors for predicting recovery of hearing loss.A retrospective, multicenter trial was conducted.Two hundred three patients examined within 7 days after the onset of ISSNHL received prednisone with lipo-prostaglandin E1. Pure-tone auditory tests were performed before and after treatment with these drugs. Blood tests were performed on blood samples collected during the patients’ initial visit to our clinic.In all patients, elevated white blood cell (WBC) counts, fasting blood sugar levels, HgbA1c, and erythrocyte sedimentation rate (ESR) significantly correlated with high hearing threshold measurements obtained on the initial visit. High fibrinogen levels, WBC counts, ESR, and low concentrations of fibrinogen degradation products (FDP) were associated with lower hearing recovery rates. Additionally, different audiogram shapes correlated with different blood test factors, indicating that different pathologies were involved.High fibrinogen levels measured within seven days after ISSNHL onset correlated with poorer hearing recovery. This may be a consequence of ischemia or infections in the inner ear. The high WBC counts also observed may therefore reflect an immune response to inner ear damage induced by ischemic changes or infections. Our data indicate that therapeutic strategies should be selected based on the timing of initial treatment relative to ISSNHL onset. Idiopathic sudden sensorineural hearing loss (ISSNHL) is characterized by a sudden onset of hearing loss and generally presents unilaterally Many studies have identified factors that predict which patients possess a favorable prognosis and require minimal or no treatment. However, only a few studies have determined the probability of whether a patient’s hearing will recover by using prognostic models that incorporate probability data ,Other researchers have tested various blood indices as potential prognostic factors for ISSNHL. Analyses of peripheral blood samples from patients who experience ISSNHL revealed they present with certain vascular risk factors, such as elevated plasma and whole blood viscosity It is well known that vascular risk factors can affect inner ear function by compromising the vascular endothelium, leading to functional disruption White blood cell (WBC) count is another potential prognostic factor. High WBC counts are frequently found in ISSNHL patients In this study, we performed hearing tests and analyzed peripheral blood samples to characterize the pathology of ISSNHL and to identify prognostic factors for predicting hearing recovery.Subjects were 203 patients with unilateral ISSNHL , who werThe study was conducted according to the principles expressed in the Declaration of Helsinki. Inclusion criteria were as follows: (1) hearing loss appeared acutely, without recognizable cause; (2) hearing loss was sudden, sensorineural in origin, and untreated; (3) patients were seen at our clinics within 7 days of onset; (4) patients followed up until their hearing recovered; (5) patients presenting with hearing loss of more than a 30 dB hearing level (HL), affecting at least three frequencies; (6) normal hearing in the contralateral ear ; (7) negative history of hearing loss or ear surgery in the affected ear; (8) lack of impairment of the cranial nerves ; and (9) gadolinium-enhanced magnetic resonance image verifying negative cranial nerve VIII pathology. All patients affected by Ménière’s disease, noise-induced hearing loss, and other known causes of inner ear disease  were excluded  Protoco.On the patients’ initial visit to one of our clinics, a complete clinical history was obtained and standard audiovestibular exams were performed. These consisted of pure-tone testing and MRI of the internal auditory canal and posterior cranial fossa. Peripheral blood samples for chemistry and hematological tests were collected from the patients after informed consent was obtained but before treatment was given. All patients were then subjected to the same treatment protocol, which consisted of intravenous prednisone (60 mg tapered to 20 mg), Lipo PGE1 (vasodilators), ATP, and vitamin B12 for 7 days. Hearing improvement was evaluated using the following improvement rate criterion described by Ogawa et al. Blood samples were analyzed. Clinical chemistry determinations were made for glucose, hemoglobin A1c (Hgb A1c), total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL), and serum triglyceride levels. Hematology determinations were made for WBCs, red blood cells (RBCs), thrombocytes, hemoglobin, hematocrit, and ESR. Hemostasis determinations were made for prothrombin time (PT %), activated partial thromboplastin time (APTT %), fibrinogen, and fibrinogen degradation products (FDPs).We examined whether initial hearing or hearing recovery rate correlated with the above-mentioned blood data values. All results were expressed as mean values ± standard deviation. Statistical analysis was performed using parametric and nonparametric tests .The data obtained from patients with ISSNHL were summarized and tabulated. High WBC counts 1–7 days after the onset of sensorineural hearing loss correlated with hearing recovery rate . In subj2 = 0.075, Adjusted R2 = 0.060). Multivariate analyses (multiple regression) failed to identify any relevant factors.We also classified several types of audiogram shapes and analyzed their correlation with prognostic factors see . We usedHere, we report results from an ongoing multicenter clinical trial evaluating prognostic factors for ISSNHL. In all 203 patients included in this study, elevated fibrinogen levels and WBC counts correlated with poorer hearing recovery rates and poorer prognosis. Higher ESR values and WBC counts obtained during the patients’ initial hospital visit significantly correlated with higher hearing thresholds (average threshold measured at five frequencies).It remains unclear whether ESR is a reliable prognostic factor for ISSNHL, as some researchers consider it to convey a poor prognosis As previously reported, ANA titer and ESR are two parameters that are significantly more elevated in ISSNHL patients than in normal individuals The elevated WBC counts in our patients may be due to an immune response to more extensive tissue damage in the inner ear. This hypothesis is consistent with our finding that higher WBC counts negatively correlates with hearing recovery rates in patients having a “total deafness” audiogram shape and in patients with severe hearing loss .Higher WBC counts did correlate with hearing level measured during the initial visit and hearing recovery of patients that sought treatment within 1–7 days after onset  and 8. MAs mentioned, within 1–7 days of ISSNHL onset, fibrinogen levels correlate negatively with hearing recovery rate, and elevated ESR correlates with the patients’ hearing level at the initial visit. The finding that fibrinogen levels measured one day after ISSNHL onset correlates with hearing recovery rate indicates that, at ISSNHL onset, blood viscosity is high and thus blood supply to affected areas is decreased. We surmised that a few days thereafter, inner ear tissue damage further progresses due to WBCs being drawn to the site by adhesion molecules expressed by local blood vessels.From the start of this study in 2005, we have used vasodilators to treat ISSHL, even though their use for this purpose remains unsubstantiated due to the poor hearing outcome. However, one of our co-authors (K. Ogawa) has observed marked hearing improvement at higher frequencies and significant improvement of tinnitus in patients receiving PGE1 compared to placebo (44% versus 13%), even though no significant differences were observed in the overall hearing gain or in the rate of hearing improvement between the PGE1 and placebo groups of patients The time course of molecular factors, such as adhesion molecules, contributing to inner ear damage might be influenced by transcriptional factors. Indeed, nuclear factor kappa B (NFkB), a transcriptional factor that activates cytokines and adhesion molecules, may be involved in inner ear damage or sensorineural hearing loss We also demonstrated that audiogram shape varies across different pathological backgrounds that require different prognostic factors for blood tests.Our data demonstrate that patients with higher LDL values have lower hearing recovery rates. High LDL and high cholesterol levels increase atherosclerosis and blood viscosity. Cholesterol, however, is not prognostic factor for ISSHL. This may induce extensive inner ear ischemia, which could be responsible for the “total deafness” audiogram pattern observed in some of our patients.Canis et al. recommended fibrinogen and LDL apheresis in patients presenting with high fibrinogen and LDL levels In the present study, we propose a novel therapeutic concept for the treatment of ISSNHL, in which treatment choice depends on the timing and type of audiogram obtained.We demonstrated that high fibrinogen levels (measured one day after ISSNHL onset) correlate with poorer hearing recovery in ISSNHL. High fibrinogen levels may indicate ischemic changes in the inner ear Table S1Data of all the patients in this study.(XLSX)Click here for additional data file.Checklist S1TREND checklist.(PDF)Click here for additional data file.Protocol S1We provided the protocol .(DOCX)Click here for additional data file.


Hearing loss from birth up to the age of 3 years has a negative effect on speech/language development and results in sensory, cognitive, emotional, and academic defects in adulthood by causing delayed development of communicative-linguistic abilities. The present study was performed in order to assess the effect of early intervention on language development in Persian children aged 6-7 years with severe sensorineural hearing loss.Thirty boys and girls aged 6-7 years participated in this study, all of them had severe congenital sensorineural hearing loss in both ears. All children were using bilateral behind-the-ear hearing aid, and had similar economic/socio-cultural backgrounds. Subjects were categorized into two groups based on the age of identification/intervention of hearing loss (3-6 and 12-15 months of age). The Persian TOLD-P3 test was used to evaluate language development in all subjects. Data collection was accomplished by observation, completion of questionnaires, and speech recording.There was a significant difference in language development in 11 sub-tests and five lingual gains on the Persian TOLD-P3 test between early (3-6 months of age) and late identified/intervened (12-15 months of age) hearing-impaired children (PEarly identification/intervention of hearing loss before the age of 6 months has a significant positive effect on a child’s language development in terms of picture/relational/oral vocabulary, grammatical comprehension, sentence combining, grammatical completion, phonologic analysis, word differentiation, word production, semantics, and syntax. Moreover, early identification/ intervention of hearing loss develops the hearing-impaired child’s lingual gains in visual vocabulary, grammatical completion, word differentiation, phonologic analysis, and word production. Verbal language perception, development, and usage is strongly related to the auditory sense. Therefore, the presence of hearing loss – even to a mild extent – has a negative effect on speech–language development in hearing-impaired children, and delays acquisition of linguistic, social, academic, and sensory abilities. Further, as speech and language development are prerequisites for cognitive development, an auditory defect may affect and impair the hearing-impaired child’s cognitive ability ,2. VerbaPre-lingual hearing loss has a negative effect on all fields of language acquisition, but the influence on phonology,morphology, advanced vocabulary, and syntax is most profound . BecauseIdentification of hearing loss and early appropriate intervention before the age of 6 months can increase the possibility of normal speech and language development in hearing-impaired children ,13. The Many studies have confirmed the significant positive effects of early identification of hearing loss on speech, language, and socio-emotional develop- ment. Murria et al. showed that hearing-impaired children who have received appropriate and early hearing aid assessment and fitting at the age of 3 months and cochlear implantation at the age of 9 months can reach normal language development in up to 96% of cases . HearingThe presence of hearing loss at critical periods of language development causes disorders in speech acoustic processing and language synthetics–syntax representation and results in defects in language acquisition and synthetics-syntax usage. Language learning in hearing-impaired children requires the presence of natural conditions; therefore, their lingual environment must be the same as for normal-hearing children. Most synthetic and syntax abilities are learned at critical periods of language development, and this is affected by different variables such as the mother’s speech, the complexity of heard sentences and repetition–communicative situations . DespiteIt is note-worthy that the development of semantic and syntax skills are the basis of academic progress in school. As school entrance age is 6–7 years in Iran, evaluating these skills before entering the school will be useful for educational/ verbal rehabilitative program planning. Despite the availability of several similar studies concerning verbal skills in Persian hearing-impaired children, there are currently no published studies on verbal skills in Persian children aged 6–7 years with hearing impairment. However, suitable rehabilitative program planning requires accurate identification of language deficiency for every hearing-impaired child, individually. This may be obtained through precise and detailed evaluation of hearing-impaired abilities in different semantic and syntax aspects by utilizing a proper and plenary tool such as TOLD-P3. Although several studies have been published relating to the effect of early intervention on language development in Persian hearing-impaired children, none have used such a test. Hence, our study is the first research into the evaluation of lingual gains in Persian hearing-impaired children, and is thus an unprecedented and innovative study of the lingual abilities of Persian children with hearing impairment. Considering the importance of language development as a principal prerequisite for socio-academic success at school, this study was performed to determine the effect of early hearing loss intervention on language development in Persian children aged 6–7 years with severe sensorineural hearing impairment before starting school.Thirty children (14 girls and 16 boys) aged 6–7 years with severe sensorineural hearing impairment participated in our study. The entry criteria were having bilateral congenital flat severe sensorineural hearing loss (70–85dB), normal tympanic membrane, tympanogram and IQ scores (based on Goodenough–Harris Test scores of 90–110), no other handicap, binaural hearing aid fitting (used for 12–14 hours per day), and the similarity of the intervention program received. All children had the same socio-cultural background and were classified into two groups depending on the age of hearing loss identification/ intervention. The first group consisted of six girls and nine boys with a hearing-loss identification/ intervention age of 3–6 months and the second group consisted of seven girls and eight boys with a hearing-loss identification/intervention age of 12–15 months. These children were selected among other children supported by the Narmak Welfare Organization Center by an available sampling method. All subjects were monolingual, right-handed children who lived with literate, normal-hearing parents. Their communicative pattern was verbal language. Safety and ethical aspects of this research project were ensured by the Iran Medicine Sciences University.The first stage of the data collection process was completion of a questionnaire consisting of questions about the individuals' medical-familiar history, age of hearing loss identification/ intervention, quality of intervention program . Next, an audiologic evaluation including otoscopy, immittance and pure tone audiometry was performed in all children. Immittance and pure tone audiometry were undertaken in the Narmak Center's audiology clinic using a Pejvak Ava ZA86 and Pejvak Ava CA86 clinical audiometer, respectively. Acoustic stimuli were delivered via TDH39 supra-aural headphones. The lingual abilities were assessed using the Persian TOLD-P3 test as well as direct observation, questionnaire completion and speech recording as performed at the end of the data collection process . Raw andThe Kolmogorov–Smirnov test showed normal distribution of the sample. An independent T–test was used to compare the means of scores in all sub-tests and lingual gains.SPSS18 used to perform statistical analysis, and the putative level of significance was defined as P<0.05.Descriptive data relating to standardized scores for picture/ relational/oral vocabulary, grammatical comprehension, sentence combining, grammatical completion, phonologic analysis, word differentiation, word production, semantics and syntax are presented in .Comparing the results obtained from these 11 sub-tests showed a statistically significant difference between lingual abilities in the two groups (P<0.05). Moreover, there was a significant difference in the standardized scores for the five lingual gains  between the early and late identification/intervention groups (P<0.05) .This study showed a significant difference between the developments of lingual abilities in children with early compared with late identified/intervened hearing loss. Lingual aspects assessed were abilities in semantics, syntax and phonology through 11 sub-tests  and five lingual gains . Ability in semantic ability was evaluated by picture/relational/oral vocabulary and semantic sub-tests. Knowledge of vocabulary, objects and events constitute semantic ability, which offers the possibility of thinking about language and talking about words, as well as their proper use. Thus, the development of semantic ability provides the basis for using a defined word for a defined function or target expression . HearingThe significant difference between syntactic skills in children with early versus late identified/intervened hearing loss was another finding that was evaluated by grammatical comprehension, sentence combining and grammatical completion of sub-tests. These differences support the negative effect of late identification of hearing loss on syntactic abilities and the importance of early appropriate hearing loss intervention in hearing-impaired children. Syntactic skills enable children to use syntactic morphemes, adverbs, prepositions, pronouns, compound sentences and verb suffixes properly. Based on their degree of hearing loss and the quality of the intervention program used, hearing-impaired children have syntactic difficulties in such a way that their syntactic construction significantly relies on putting disjointed single words together. This phenomenon is not seen in the lingual construction of normal-hearing children . Improvement in phonologic skills was assessed by word production, word differentiation and phonologic analysis sub-tests in the both early and late identified/intervened hearing-loss children. There was a significant difference between phonologic skills in the two groups; early identified/intervened impaired-hearing children showed the positive effect of early appropriate intervention on phonologic skills development in hearing-impaired children. Phonologic skills support the ability of analyzing words into their phonologic elements and reading/ writing development in school. Moreover, control of produced speech pitch-loudness-rate and perception of heard speech pitch-loudness-rate results from phonologic skills development. Unsuitable usage of phonemes, word onset/offset consonant omission and a known vowel addition between two neighboring vowels are common phonologic disorders in hearing-impaired children that decreases the clarity of produced speech, especially in complex conversation backgrounds. Stress disorders result from inappropriate usage of phonetic duration, breathing control weakness, repetitious pauses in the speech continuum, breathing-speech producing imbalance, speech tonality disorder, and abnormal speech rhythm and enumerate as the most common speech abnormalities in hearing-impaired children. Identification/ intervention of hearing loss before the age of 6 months provides the possibility of language acquisition in hearing-impaired children in the same way as same as normal-hearing children, and reduces lingual abnormalities in these children .Another finding of this study was a significant difference between the mean standardized scores of the combined lingual gains in the two assessed groups, which showed that children with early identified/intervened hearing loss have more prominent combined lingual gains than late identified/intervened children. The applied lingual gains were visual vocabulary, grammatical completion, word differentiation, word production, and phonologic analysis aspects. Lingual gains are predictive indicators for a child’s reading and writing abilities at school . TherefoNo significant difference was seen between girls and boys in the lingual abilities assessed, showing that there is no effect of gender on language development of hearing-impaired children if they receive appropriate intervention. Hence, it seems that the effects of gender on improvement in lingual ability are seen only at the beginning of language acquisition .Our findings were also consistent with those of Yoshinaga-Itano (2003) who showed that early identification/ intervention of hearing loss before the age of 6 months enables normal lingual/ cognitive development in hearing-impaired children regardless of their degree of hearing loss, gender, race, socioeconomic level and communicative methods. His study also revealed that children with early identified/intervened hearing loss have higher expressive language scores . In 1998Based on the results of our study, the importance of early identification/ intervention of hearing loss is supported. Recent technology has made it easier to identify/ intervene in hearing loss at a younger age. Earlier studies have shown that the mean age of hearing-loss intervention in Persian hearing-impaired children was 3–6 years and found it has more recently been reduced to 2.5 years . Now, thBecause the relationship between lingual disorders and reading/writing disabilities is evident , it is pThe present study supports the proficiency of the Persian TOLD-P3 test for perceptive/expressive language evaluation in Persian children with severe hearing impairment. Moreover, there was a significant difference in the synthetic and syntax skills of severely hearing-impaired Persian children with early versus late identification/intervention.Because synthetic and syntax skills are the basis for academic progress in school, so assessment of lingual development in hearing-impaired children will help in assessing lingual deficiencies and planning adequate auditory/verbal rehabilitative provision prior to entering school. Therefore, it is necessary to reduce the age of identification/ intervention of hearing impairment to 6 months or earlier in order to promote normal lingual development and proper emotional, academic, social and sensory growth.


The patterns of viral location and the tissue distribution of CD4- and CD8α-positive cells were concurrently detected by immunohistochemical staining, which revealed respiratory and digestive organs as the primary sites of antigen-positive signals. Average AIV H9N2 viral loads were detected in the feces, Harderian gland (HG), and trachea, where higher copy numbers were detected compared with the rectum. Our results suggested the strong induction of proinflammatory cytokine expression compared with interferons (IFNs). Notably, in most tissues from the AIV H9N2 strain-infected birds, IFNα and IFNγ gene transcripts were differentially expressed. However, inverse changes in IFNα and IFNγ expression after AIV H9N2 strain infection were observed in vitro. Taken together, the results suggest that AIV H9N2 is widely distributed in multiple tissues, efficiently induces inflammatory cytokines in the HG and spleen of goslings and inversely influences type I and II IFN expression both in vivo and in vitro. The findings of this study further our understanding of host defense mechanisms and the pathogenesis of the H9N2 influenza virus in geese.Geese, as aquatic birds, are an important natural reservoir of avian influenza virus (AIV). To characterize the innate antiviral immune response against AIV H9N2 strain infection in geese as well as the probable relationship between the expression of immune-related genes and the distribution of viral antigens, we investigated the levels of immune-related gene transcription both in AIV H9N2 strain-infected geese and  The virus stocks were propagated in specific pathogen-free (SPF) chicken eggs, and the copy number for AIV was measured to be 7.14 × 1050: 10–7.375/0.2 ml). Five PBS-treated goslings served as controls. After infection, the challenged goslings were monitored daily for clinical signs of disease, and their body weights were measured. The two groups of goslings were maintained in different isolation units in a biosecure animal building and fed a commercial diet ad libitum. Both the PBS-treated and the virus-infected goslings  were sacrificed at 5 days post-infection (5 dpi). Different tissues, including the T, SP, BF, CT, HG, SI, P, R, LU, TR, and LI, were collected. The animal studies were approved by the Institutional Animal Care and Use Committee of Sichuan Agricultural University and followed the National Institutes of Health guidelines for the performance of animal experiments.The study was conducted with 10 three-day-old AIV-free (confirmed by PCR detection) Sichuan White goslings. Five goslings each were infected AIV H9N2 by oral administration (250 μl) or intranasal injection (250 μl)  (TCID50: 10–7.375/0.2 ml) were then added to each well. The final concentration of poly IC (30 μg/ml) agonist (positive control) was added into medium. The cells were harvested for total RNA isolation at 6 h after stimulation, with PBS-treated wells serving as controls.Peripheral blood mononuclear cells were isolated and purified from healthy adult geese. The PBMCs were grown overnight in RPMI supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C and 5% COTotal RNA was isolated from selected tissues using RNAiso Plus reagent . The quantity and integrity of the RNA in each sample were determined using a NanoDrop 2000 . Complementary DNA (cDNA) was synthesized using a 5X All-In-One RT MasterMix Reagent Kit in accordance with the manufacturer’s instructions . Finally, the samples were stored at –80°C until use.Table 1. Real-time PCR was performed using the Bio-Rad CFX96 Real-Time Detection System . Target gene expression was normalized to β-actin, a constitutively expressed endogenous control gene. Real-time PCR conditions and protocols were carried out as previously described . The expression of goose immune-related genes was analyzed and selected based on specificity, as determined according to dissociation curves. The primer sequences are listed in escribed .–1 to 10–8 to establish the standard curve. The primer sequences for the M gene used in the present analysis are given in Table 1. The program parameters consisted of initial denaturation at 95°C for 10 min, followed by 40 cycles at 95°C for 15 s and 60°C for 1 min. A melting curve analysis was performed at the end of the reaction to assess the specificity of the PCR amplification. cDNA was synthesized from the RNA sample using the Uni12M primer. The cDNA from the cloacal feces and the viral cDNA from tissues were used as templates for detection, and each sample was analyzed in triplicate.To examine the shedding titre in cloacal swabs in the later part of the digestive system, the M gene of AIV H9N2 was amplified and then subcloned into the pMD18T vector  to prepare the reference DNA templates for qRT-PCR. The recombinant plasmid was serially diluted from 10t-test. A P value less than 0.05 was considered to be statistically significant.Data are indicated as the mean and standard deviation. The statistical analysis was performed on single and paired samples, as appropriate, by applying Student’s Figures 1A,E); cellular swelling, congestion, and hepatic sinusoid disappearance ; suffuse hemorrhage in the SP ; and moderate pathological manifestation in the SI .In this study, we carried out infection with an LPAIV strain [A/chicken/JS/C1/2008 (AIV H9N2)]. The AIV H9N2-infected goslings did not show any obvious difference in weight gain or daily feeding at 5 dpi compared with the control group. The AIV H9N2-infected goslings showed slight weakness, a loss of appetite, and loose stool excretion. Several of the goslings like to squat together, whereas other goslings were unwilling to stand and remained close to their counterparts. A histopathological analysis showed obvious pulmonary hyperaemia and congestion . The AIV antigen was mostly strongly distributed in the SP (+++) , SI (+++) , R (+++)  and lung (+++) ; was moderately detected in the BF (++)  and the LI (++) ; and was faintly detected in the TR (+) . Importantly, the antigen was readily and widely distributed in the mucous membrane epithelium and myofibrocytes of the intestines (+++)  and in the follicle-associated epithelium, corticomedullary arch-forming cells, and epithelial reticular cells of the BF (++)  as well as in the pseudostratified columnar epithelium of the TR (+) . In goslings 5 dpi, CD8α-positive cells were found scattered throughout the SP (+++)  and were most prevalent in the medulla and epithelial reticular cells of the BF (++) , the hepatocytes in the LI (++) , the pseudostratified columnar epithelium of the TR (+)  as well as in the SI (+) . These cells were also faintly detected in the LU  and in the villi of the R (+) . Moreover, CD4-positive cells could be detected in the mucous membrane epithelium of the SI (+)  and in the hepatocytes and mesenchymal cells of the LI (++)  and were faintly detected in the SP , LU , R , TR  and BF (+) .The distribution and intensity of the immunoreactivity for the AIV antigen in paraformaldehyde-fixed, paraffin-embedded tissue sections after infection, as determined by IHC, are shown in P < 0.01) and SP. A negative trend in other immune-related tissues was found, especially in the BF (P < 0.05) . Moreover, the IFNα was repressed in the BF at 7 dpi, but not at 1 and 3 dpi . In other tissues, there was no significant difference in IFNα and IFNγ expression, but a negative trend for these IFNs was generally observed in the LI . Goose IL1β and IL6 gene expression was dramatically reduced in CT (P < 0.01); these interleukins were moderately expressed in the LI and LU and minimally expressed in the SI and T of infected goslings. As observed in Figure 3, IL6 was strongly upregulated in the HG (P < 0.01), SP (P < 0.01), and TR (P < 0.01) compared with the controls. The expression of goose MyD88 was inhibited to varying degrees in both primary immune tissues and other tissues.Inconsistent changes in immune effector expression were detected in various goose tissues. In the selected immune-related tissues (except the SP), IFNα gene expression was relatively lower in the AIV H9N2-infected group, whereas IFNγ transcript expression was increased in the HG (P < 0.01) and IFN-γ (P < 0.01), IL-1β (P < 0.01), IL-6 (P < 0.01), as well as MyD88 (P < 0.01) . In the AIV H9N2-infected goose PBMCs , elevated levels of IFNα and IFNγ were found after 6 h of stimulation . Proinflammatory cytokine transcripts, including IL1β and IL6, were significantly upregulated at 5 dpi . Consistent with this, the MyD88 transcript was also significantly upregulated in the infected cells (P < 0.05).The mRNA transcript levels of goose interferons and pro-inflammatory cytokines were measured to quantify the response of goose PBMCs to poly IC (30 μg/mL). After 6 h incubation, poly IC induced a significant up-regulation of goose IFN-α , feces (107.36 copies/μg), HG (106.74 copies/μg), TR (106.24 copies/μg), and CT (105.64 copies/μg) .Faeces were collected from the intestinal tracts of geese, and peripheral blood and tissues, including the HG, TR, CT, T, BF, and R, were collected. The viral copy number was then detected by qRT-PCR. The efficiency of M gene amplification was 100.1%, and Large pools of AIV cover a variety of subtypes of wild water birds, including wild ducks and geese. The prevention and treatment of AIV remain a challenge because of our incomplete understanding of the restriction and dissemination of AIV in aquatic birds as well as the lack of comprehensive information regarding innate immune regulation in birds. Although the pathogenesis of AIV in geese has not been fully elucidated, there is evidence that water birds act as mixed reservoirs of currently circulating and pervasive avian influenza strains. Specifically, certain avian H9N2 viruses harbor receptor-binding characteristics similar to those of human strains of the virus, which are a contributing factor to increases in both the potential reassortment of AIV and potential transmission between diverse species .in vivo may have correlated with viral loads; therefore, AIV H9N2 may persist in gosling to evade the IFN response.The results of our study suggest that during the early infection phase, geese showed moderate clinical symptoms, with no appetite and moderate diarrhea. The mechanisms accounting for how LPAIV H9N2 strains infect avians remain to be explored. Notably, geese are natural reservoirs of AIV and can survive after infection by AIV strains without obvious clinical signs. The differences in immune-related gene expression between duck and chicken PBMCs infected by AIV H11N9 are characterized by asymptomatic and lasting infections in ducks and rapid clearance in chickens . Our stuin vitro. The expression of IFNα observed in the in vivo study was not confirmed in the H9N2 virus-infected cells; that is, the H9N2 strain did not behave similarly with respect to IFN expression in vivo and in vitro. It appears that the host response against this strain may vary depending on the type of host cell. Additionally, these findings reflect the complex pathogenesis of AIV. There was also no significant change in IFN gene expression levels in certain tissues in vivo post-infection with the H9N2 virus. The reasons for this may be that the cytokines were transiently expressed and that the samples differed between individuals. In our studies, IFNα was suppressed to a certain extent in vivo, which was in accordance with the fact that AIV can inhibit IFN-mediated antiviral responses  showed an upward trend in certain tissues. Thus, the host may become more susceptible to secondary infection by other pathogens due to the inhibition of IFN induction. This may be the underlying explanation for the substantial innate immune system changes observed in geese after AIV H9N2 infection and propagation. These findings suggest that certain specific subtypes of the AIV H9N2 virus may participate in the negative modulation of the host adaptive immune response in aquatic birds. Further details of the real-life complex mechanisms of host defense and the immune responses of AIV H9N2-infected geese should be elucidated.Toll-like receptors (TLRs) can function as sensors to safeguard the body from danger signals. Viral recognition relies on PRRs; for example, the ligands for TLR3 are dsRNAs derived from viruses, and the ligands for TLR7 are ssRNAs derived from RNA viruses. TLR recognition activates downstream signal transduction to activate nuclear factor (NF)-κB or IFN regulatory factor (IRF) 3/7, which ultimately induces IFN and inflammatory cytokine production . AIV mayesponses . One hypesponses . The NS1activity . Althougin vivo, which is different from the cytokine expression in vitro. The results of this study not only have addressed AIV H9N2 antigen distribution in geese but also have expanded our fundamental understanding of the goose immune response against AIV H9N2 infection.In our study, LPAIV H9N2 infection in aquatic birds resulted in obscure superficial clinical syndromes but a wide antigen distribution, causing respiratory, and digestive dysfunction. Extensive virus replication was primarily observed in the goose respiratory system and intestinal tissues. In particular, the SP, SI, R, LUs, and peripheral blood all showed high viral copy numbers, indicating lasting and asymptomatic infection in the geese. Furthermore, goose IFNs were differentially suppressed by AIV H9N2 infection ZH performed most of the experiments with the help of YB and CS; ZH, WM, JR, and ZD performed the data analysis; CS and CA designed the study; WM, JR, ZD, LM, LF, YQ, WY, SK, CS, and CA supervised the study and provide the materials. ZH and CS wrote the paper. CH, CX, and JB provided the viral strain and gave the experimental support.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


NLRP3,primarily in exon 3, leading to the autoinflammatory symptoms observed in CAPS. Here, we describe a family with autosomal dominantly inherited, unilateral sensorineural hearing loss, found to have a novel mutation in exon 7 of NLRP3.Cryopyrin-associated periodic syndromes (CAPS) are a spectrum of autoinflammatory disesase with spontaneous activation of the NLRP3 inflammasome, leading to hypersecretion of IL-1b. To date, more than 90 mutations have been described in To evaluate the clinical and genetic features of a 2-generation pedigree with unilateral sensorineural hearing loss.NLRP3 was performed.Patient data, including audiologic studies, and detailed family history was obtained. DNA was isolated using saliva samples from all family members and Sanger sequencing of all 9 exons of NLRP3 exon 7: c.2753 G>A, R918Q in all 4 affected members of this 5-person pedigree. Modeling of the mutation localizes the substituted amino acid residue to the inner surface of the LRR domain and suggests an alteration in charge distribution predicted to affect inter or intraprotein protein-protein interactions.The proband was first identified during an evaluation for recurrent fevers and aphthous stomatitis, occurring 2-3 times per month, beginning at 4 months of age, without infectious etiology. Episodes were not associated with cold exposures or rash. Physical exam was notable for an otherwise well appearing child, without evidence for inflammation or autoantibodies. At the age of 5, he was noted to have unilateral sensorineural hearing loss in the range of 4000-8000 Hz, similar to his father. Two younger siblings were noted to have similar symptoms, with similar age of onset, including unilateral hearing loss. Between flares, ESR and high sensitivity CRP were normal in all three children suggesting an absence of chronic inflammation. We identified a novel variant in NLRP3.While bilateral sensorineural hearing loss has been described in association with Muckle Wells Syndrome and Neonatal onset multi-inflammatory disease (NOMID), both on the CAPS spectrum, in this family, a novel NLRP3 variant was associated with unilateral hearing loss in the absence of serologic evidence for chronic inflammation. This is the first potentially disease associated variant to be described in exon 7 of


The use of etomidate is a risk factor for relative adrenal insufficiency in patients undergoing cardiopulmonary bypass (CPB) . The objA prospective cohort study on CPB patients who received etomidate or not during anesthetic induction during 2008 to 2011. Demographic and postoperative variables were collected. We tested the Hardy-Weinberg equilibrium in order to avoid selection bias. V18 SPSS was used.2: 0.6; P = 0.42). A total of 254 (58.7%) patients received etomidate, 152 out of them required vasoactive drugs. Homozygous G was defined as unfavorable TNFβ versus the A allele [F: 18; P < 0.001): 1,849  versus 2,493  dinas.seg/cm5.m2, the presence of homozygous G , and also showed a significant etomidate-homozygous G interaction (F: 22.8: P < 0.001): 1,687  versus 3,041  dinas seg/cm5. m2  men and 148 (34.2%) women, 66 ± 6 years, EuroSCORE I 5.3 ± 4%. TNFβ was in Hardy-Weinberg equilibrium (χEtomidate use is associated with lower postoperative SVRI which is increased in the presence of G homozygosity for TNFβ polymorphism.


Moreover, active regulation of heat transfer from the beak has recently been demonstrated in a toucan , with the large beak acting as an important contributor to heat dissipation. We hypothesised that hornbills (Bucerotidae) likewise use their large beaks for non-evaporative heat dissipation, and used thermal imaging to quantify heat exchange over a range of air temperatures in eighteen desert-living Southern Yellow-billed Hornbills (Tockus leucomelas). We found that hornbills dissipate heat via the beak at air temperatures between 30.7°C and 41.4°C. The difference between beak surface and environmental temperatures abruptly increased when air temperature was within ~10°C below body temperature, indicating active regulation of heat loss. Maximum observed heat loss via the beak was 19.9% of total non-evaporative heat loss across the body surface. Heat loss per unit surface area via the beak more than doubled at Ta > 30.7°C compared to Ta < 30.7°C and at its peak dissipated 25.1 W m-2. Maximum heat flux rate across the beak of toucans under comparable convective conditions was calculated to be as high as 61.4 W m-2. The threshold air temperature at which toucans vasodilated their beak was lower than that of the hornbills, and thus had a larger potential for heat loss at lower air temperatures. Respiratory cooling (panting) thresholds were also lower in toucans compared to hornbills. Both beak vasodilation and panting threshold temperatures are potentially explained by differences in acclimation to environmental conditions and in the efficiency of evaporative cooling under differing environmental conditions. We speculate that non-evaporative heat dissipation may be a particularly important mechanism for animals inhabiting humid regions, such as toucans, and less critical for animals residing in more arid conditions, such as Southern Yellow-billed Hornbills. Alternatively, differences in beak morphology and hardness enforced by different diets may affect the capacity of birds to use the beak for non-evaporative heat loss.Beaks are increasingly recognised as important contributors to avian thermoregulation. Several studies supporting Allen’s rule demonstrate how beak size is under strong selection related to latitude and/or air temperature (T Ramphastos toco, are able to vasodilate their extremely large beaks depending on thermal conditions, allowing heat to be dissipated from the beak. In this species, non-evaporative heat loss via the beak averages 60% of total non-evaporative heat loss at air temperatures (Ta) above 28°C [b), vasodilation of the networks below the rhamphotheca (the sheath of keratin that forms the outer surface of the beak), cause an increase in beak surface temperature [There is increasing evidence for the importance of beaks in avian thermoregulation , with thove 28°C . At air perature , where aa exceeds Tb [Tockus leucomelas Lichtenstein 1842) inhabiting the Kalahari Desert may be under strong selective pressure to conserve water, particularly during the summer breeding season when air temperatures are high. Breeding female Southern Yellow-billed Hornbills are confined within a nest cavity to care for the offspring, while males are entirely responsible for provisioning the female and chicks [Hornbills (Bucerotiformes: Bucerotidae) are widespread in the Afrotropical and Indomalayan regions, with members of this taxon occupying habitats ranging from arid savannas to humid tropical forests . Like toceeds Tb . Non-evad chicks . Male anWe investigated whether the beak of this Afrotropical hornbill is functionally similar in terms of heat exchange capacity to that of the Toco Toucan . FollowiThe methods used in this study were approved by the Science Faculty Animal Ethics Committee of the University of Cape Town (protocol number 2013/V24/PR). The study was carried out on private land (Leeupan Guest Farm) with permission of the landowners and of the Northern Cape Department of Environment and Nature Conservation of South Africa (permit number 1166/2013). All bird handling was done by experienced individuals.Southern Yellow-billed Hornbills are socially monogamous and widespread in southern Africa, where they occupy savanna and woodland habitats . The indZophobas morio, and transported in cotton bags to a field laboratory within 4 km of all capture sites. Morphometric measurements were taken from each individual including body mass, wing length, tarsus length, culmen length and maximum culmen height. A lateral-view photograph of the beak and body taken with a DSLR camera  was used to calculate surface areas using ImageJ™, image analysis software .In the early austral summer of October 2013, shortly prior to the breeding season, nine adult male and nine adult female hornbills were captured with spring traps (53 x 53 cm) baited with super worms, a within the cabinet was regulated using a temperature-controlled water bath circulating water through 22-mm diameter copper tubing mounted on the inner wall of the cabinet  at the bottom of the cabinet prevented increases in humidity via exhaled water vapour and was replaced before becoming saturated. A smaller chamber made of corrugated plastic with an open front and a lid on top (350 x 120 x 350 mm), was placed within the temperature-controlled chamber. Hornbills were placed individually on a perch within the smaller chamber during data collection and would generally remain in this position. Thin nylon netting  covered the open front of the smaller chamber to prevent the hornbill from moving outside of the field of view of the thermal imaging camera, or coming into contact with the copper piping or silica gel or otherwise injuring themselves. Prior to each experiment, each individual spent 30 minutes habituating to the experimental setup at the initial temperature of 15°C. The Ta in the cabinet was increased from 15 to 45°C and held stable at four set point temperatures . The Ta was considered stable when it remained within 2°C of the experimental setpoint for 10 minutes or more. The mean heating rate between each pair of setpoint Ta values was 0.7 ± 0.1°C min-1. Increasing the temperature in a ramped fashion minimised the time each bird spent in captivity. The Ta values we used are within the range that birds naturally encounter in the wild at the study site.Birds were individually subjected to a ramped profile of increasing air temperatures inside a darkened, custom-built temperature-controlled chamber (1200 x 400 x 300 mm), constructed of corrugated plastic insulated with polystyrene (30 mm thickness). The Tn et al. ). Air mi-1. The Ta in the chamber was monitored with a NiCr-NiAl thermocouple  at 5-min intervals, and Ta and relative humidity were also recorded with three Thermochron iButtons  at 1-min intervals. The iButtons were calibrated in a circulating water bath against a factory-calibrated NiCr-NiAl thermocouple . Water vapour pressures (WVP) increased with 0.0289 kPa per 1°C air temperature increment as a consequence of the bird being present in the chamber  at a frame rate of 15 frames s chamber . Howeverb of each hornbill was measured at the start and end of each experiment to assess whether any individuals became hyperthermic during trials. A fine-gauge NiCr-NiAl thermocouple  was inserted approximately 10 mm into the cloaca, a depth at which a slight withdrawal did not result in a change in the measured Tb value. Three males and one female were removed from the chamber early as they became restless at Ta < 35°C, giving final sample sizes of 6 males and 8 females at Ta > 35°C. All individuals were released at the site of capture immediately after completion of the experiment.The onset of panting in the hornbills was visually assessed from the recorded thermographic image sequence. Ta during the course of the experiment. For each individual, one thermal image per 2.5°C Ta increment from 15°C to 45°C was sampled for surface temperature analysis. Preliminary analyses of these images revealed that beak surface temperature typically changed rapidly above a threshold Ta value. We averaged the Ta where the difference between beak temperature and Ta was greatest for the lower mandible, and total beak, for each individual in order to identify the threshold air temperature at which this change occurred (threshold Ta). Differences in panting and beak threshold Ta in response to air temperature between males and females was assessed with a Welch two sample t-test [We assessed how much each region of the hornbill body contributed to overall non-evaporative heat exchange at different TSurface temperature analysis was done by manually selecting the area of the torso, the gular skin, the lower mandible of the beak and the entire beak in each thermal image using ThermaCAM Researcher Pro 2.9 software . The polygon function in this software allows for accurate selection of the body part of interest and exports the minimum, maximum and mean temperature and the standard deviation of the surface temperature of the selected area. The feet were excluded from this analysis since they were not always visible in the thermal images.s, component parts: torso, ‘Ts_torso’`bare gular skin, ‘Ts_skin’; beak, ‘Ts_beak’) and air temperature (Ts-Ta) were calculated for the different regions of interest across the Ta gradient. For all Ts except Ts_beak, we modelled Ts-Ta data using linear mixed models with Gaussian error distribution, Ta as the predictor variable and hornbill identity as a random factor. We split the Ts_beak -Ta dataset below and above the Ts_beak threshold Tas (lower beak and whole beak) and fitted linear mixed models with Gaussian error distribution to each of the two subsets, again with hornbill identity as a random factor, and Ta as the predictor. Linear mixed models were fitted by REML in R 3.1.2 [Differences between average body surface temperature  below and above the beak threshold Ta. This was done in order to be able to assess the fractional contribution of each body region to total heat exchange, taking into account convective and radiative heat exchange  were not included in this analysis, because the Ts_beak at this stage was cooler than Ta, resulting in a negative flux value. At these high Ta values, the hornbills were observed to pant, indicative of a switch to evaporative water loss as the primary mode of heat dissipation. Linear mixed models with Gaussian error structure were fitted by REML to assess the capacity for heat dissipation in response to Ta using package lme4 [The contributions of the different body regions to total heat dissipation were expressed as mean heat dissipation , mean percentage of the total body heat dissipation, and relative heat dissipation  belowage lme4 , with inage lme4 ,18.a), the surface temperature of the beak clearly changed in response to Ta (hereafter referred to as threshold Ta), indicated by a rapid change in the colour of the beak in 14 of the 18 study individuals, as visualised by the thermal imaging camera . We found no difference in Ts_beak values between males and females at the threshold temperature . Body temperature (Tb) measurements confirmed that none of the study individuals became severely hyperthermic, with mean Tb = 41.4 ± 0.2°C before and 42.2 ± 0.2°C after the experiment.Beak surface area is a sexually dimorphic trait in Southern Yellow-billed Hornbills  and backs_beak and Ta differed markedly from that between Ts_skin and Ta and Ts_torso and Ta. As Ta increased from 15°C to 45°C, the difference between skin surface temperature and air temperature (Ts_skin-Ta) and the difference between torso surface temperature and air temperature (Ts_torso-Ta) decreased linearly . The rate of change in Ts_torso-Ta with increasing Ta was much shallower (-0.11°C per 1°C increase in Ta); the maximum difference between torso surface temperature and air temperature was 3.9 ± 0.2°C at the lowest experimental temperature (~15°C), likely due to the insulating properties of the feathers . Below the Ts_beak threshold temperature, Ts_beak -Ta increased at a rate of 0.062°C per 1°C increase in Ta and at a rate of 0.002 W per 1°C increase in Ta (s_beak -Ta threshold Ta (~30.7°C), Ts_beak -Ta was maximised and heat was radiated from the beak to the cooler environment with greatest efficiency. As Ta increased above this threshold, Ts_beak -Ta declined at a rate of 0.24°C per 1°C increase in Ta. At the threshold temperature mean Ts_beak -Ta was 3.8 ± 0.6°C and the mean heat dissipation from the beak was 0.1 ± 0.0 W: equivalent to 25.1 W m-2  compared to Ta < 30.7°C (a = 32.2°C (range 18.0–39.4°C)  . The maxOur data confirm that hornbills, like other birds, can regulate heat exchange from their beaks, using them as thermal radiators when air temperatures are high, but restricting heat loss during cold ambient conditions. The large beaks of both hornbills and toucans are highly vascularised, and control of blood is regulated by vasoconstriction and vasodilation processes . Bird bea of 33.0°C in one individual). We calculated maximum rate per unit surface area under comparable convective conditions using data presented in the figures in the paper by Tattersall et al. [-2 via their beaks, whereas the Yellow-billed Hornbills we studied only dissipated a maximum of 25.1 W m-2 via the beak. We suggest three possible explanations for these differences in heat exchange capacity in hornbills and toucans. First, toucans allowed beak surface temperature to rise at considerably cooler air temperature than hornbills (20–25°C compared to 30.5–31°C), allowing them to achieve a steeper gradient between beak surface temperature and air temperature than hornbills, which resulted in a greater capacity for heat dissipation. This difference in threshold temperature may not be an immutable species-specific response. We expect the threshold temperature for vascular recruitment to be phenotypically plastic among populations in response to acclimation to environmental conditions. Second, the rhamphotheca structure of the hornbill beak is twice as hard as that of the toucan beak [a priori, be expected to be under stronger positive selection in humid habitats. This leads to the prediction that thermal radiators such as beaks are more important for species inhabiting humid environments compared to those living in arid areas, an idea first proposed by Greenberg et al. [Although both hornbills and toucans have the ability to regulate heat exchange through their large beaks, the threshold temperature for vascular recruitment and relative beak size differed markedly between the two species. Toco Toucans were able to dissipate on average 60% of total heat loss via the beak . The heal et al.  and founcan beak , potentican beak , and is can beak . Third, can beak  predictscan beak ,39, withg et al.  and hornGallus gallus domesticus) in response to a minor discomfort trigger [In both our study and the study of toucans by Tattersall et al. , a few i trigger .a exceeds Tb. We argue that the ability of birds to vasoconstrict the beak at Ta > Tb would be beneficial to avoid warming up of the blood in the beak, but we were unable to confirm this using thermal imaging data.Our data add to a growing body of literature revealing the importance of the avian beak in thermoregulation –4,6,7,26S1 DataNumerical data used in preparation of Figs (XLSX)Click here for additional data file.S1 Figa) of torso, gular skin, the beak and lower mandible of the beak in Southern Yellow-billed Hornbills (Tockus leucomelas). Error bars represent SE. Note that the scale of the y-axis of the graphs of gular skin, total beak and lower mandible is different from graph representing the torso, this was done to better illustrate patterns of heat loss by the beak.Heat loss (Watts) plotted against air temperature (T(TIF)Click here for additional data file.S2 Figa) of torso, gular skin, the beak as a whole and lower mandible of the beak in Southern Yellow-billed Hornbills (Tockus leucomelas). Data above the panting initiation temperature (Ta = 37.4 ± 2.1°C) has not been included in this graph since evaporative heat loss has not been assessed and this makes total heat loss after initiation of panting incomplete.Heat loss as a proportion of total body heat loss (%) plotted against air temperature (T(TIF)Click here for additional data file.S3 FigRelative humidity (%) and water vapour pressure (kPa) in the temperature cabinet in response to air temperature (°C). Data are combined from all the individual experiments. Error bars represent SE.(TIF)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.S1 VideoTockus leucomelas) during the experiment.Thermal imaging sequence of the Southern Yellow-billed Hornbill ((MP4)Click here for additional data file.