Mutations in the connexin 26 gene seem to be extremely common in non-syndromic hereditary deafness genesis, especially the 35delG, but there are still only a few studies that describe the audiometric characteristics of patients with these mutations.to analyze the audiometric characteristics of patients with mutations in the connexin 26 gene in order to outline genotype-phenotype correlation.Experiment Design: Retrospective, cross-sectional study.Tonal audiometries of 33 index cases of non-syndromic sensorineural hearing loss were evaluated and eight affected relatives. Specific molecular tests were carried out to analyze mutations in the connexin 26 gene. A 27.3% prevalence of mutation 35delG was found in the index cases and 12.5% among the relatives affected. In relation to hearing loss degree, 41.5% of the patients were found with profound hearing loss, 39% with severe HL and 19.5% with moderate HL with homozygote and heterozygote patients for the 35delG predominating in the severe-moderate hearing losses.Our results suggest that the audiometric data associated with the molecular diagnose of hearing loss helped us to outline a genotype-phenotype correlation in ten patients with 35delG mutation. However, it is still necessary to run multicentric studies to verify the real phenotypic expression in the Brazilian population, as far as the 35delG mutation is concerned. It is well known that in developed countries one in 750 births is likely to produce a child with sensorineural hearing impairment and it is estimated that one in 1000 children is affected by severe deafness at birth or by the end of the prelingual periodIn terms of phenotype, the autosomal recessive forms are more severe, being responsible for almost all forms of congenital deafness. These, in turn, are caused in great part by cochlear defects, leading to sensorineural hearing impairment. The autosomal dominant forms seem to contribute more to cases of postlingual deafness, which are usually progressive, and the impairment is, in most cases, conductive or mixed Mutations in the conexin 26 or GJB2 - Gap Junction Protein Beta 2 gene, located on the long arm of chromosome 13 (13q11-12), appear to be extremely common in the genesis of inherited non-syndromic deafness, and account for 34% to 50% of autosomal recessive sensorineural deafness (DFNB1) and for 10% to 37% of sporadic casesDeletion of a guanine out of a sequence of six guanines going from position 30 to position 35 in the conexin 26 (GJB2) gene is the mutation that occurs in 60% to 80% of cases. This nucleotide deletion can occur at position 35 (35delG) or at position 30 (30delG) of the gene, but the deletion at position 35 (35delG), genetically related to chromosome 13, is the more frequent one, varying between approximately 50% to 85% of the non-syndromic deafness cases in patients from Italy, Spain, and IsraelEven though the GJB2 gene is of great importance in the etiology of non-syndromic sensorineural impairment, there are so far very few studies describing the audiometric characteristics of patients with mutations in the GJB2 gene associated with hearing loss, especially in Brazil. According to these studies, in patients who are homozygous for mutations in the GJB2 gene and mainly in those who are homozygous for 35delG, the hearing loss is characterized for being prelingual, affecting all frequencies, being non-progressive, varying in degree from moderate to deep, even among impaired siblings of the same family, and not being associated with vestibular alterations and radiological abnormalities of the inner earDuring the period from March through June, 2000, a cross-section study was carried out, in which 33 index cases  with non-syndromic sensorineural hearing impairment were investigated. They aged between 3 and 37 years, were randomly selected at the Ear-Nose-Throat Outpatient Clinic. Of these index cases, eight relatives were evaluated , aged 11 to 45 years, who also presented non-syndromic sensorineural hearing impairment. Hence, we evaluated 33 families with at least one member with hearing loss, totaling 41 impaired individuals. This study was approved by the Research Ethics Committee, Protocol No. 4429/2000. The complete history of each patient was obtained to investigate age at onset of the hearing impairment, presence of other cases in the family, and to exclude the possibility of environmental causes such as, maternal-fetal infections, perinatal complications, meningitis, use of ototoxic drugs, acoustic trauma. Physical, otorhinolaryngological and systemic examinations, as well as complementary tests, were carried out to exclude signs suggesting syndromic forms of hearing loss . Moreover, patients were submitted to ophthalmologic evaluation (including fundoscopy), vestibular tests and computerized tomography of the temporal bone. Thus, a complete clinical evaluation was performed to exclude patients with hearing loss caused by environmental factors, congenital malformations of the inner ear or genetic syndromes. The patients were audiologically tested by pure-tone audiometry, performed at the Speech Therapy Outpatient Clinic, and those with non-syndromic sensorineural hearing impairment classified as mild (25-40 dB), moderate (41-60 dB), severe (61-80 dB) or profound (>81 dB) were includedMolecular analysis was carried out at the Molecular Biology Center, after DNA extraction from whole blood, done at the Genetics and Molecular Biology Research Unit, using a genomic DNA extraction kit , according to the manufacturer protocol. To detect the 35delG mutation, the technique used was allele-specific polymerase chain reaction (AS-PCR) with specific primers8. Primers named control A (direct) and B (reverse) were also synthesized, for co-amplification of the GJB2 gene with a segment of the amelogenin gene homologous to the X-Y chromosome, thus being used as internal amplification controls19. Additionally, the PCR technique was used to detect the Delta (GJB6 - D13S1830) mutation, with specific primers20, in heterozygous samples and in those who did not present the 35delG mutation. The AS-PCR and PCR products were analyzed by electrophoresis on 1.5% agarose gel in TBE 1X buffer, containing ethidium bromide, at a concentration of 0.5mg/mL, submitted to ultraviolet light to confirm that the reaction was successful, and the gel was photographed. The samples which did not present the mutations under study on both alleles, or heterozygous samples, were submitted to automated sequencing.Two pairs of primers were synthesizedThe present retrospective study was submitted to and approved by the Research Ethics Committee, Protocol No. 2813/2004, in order to review the molecular and audiometric data of patients.Percentages with their respective standard deviations were calculated, and the results are expressed in % (SD%).Therefore, we found a prevalence of 27.3% (SD%=7.7) for the 35delG mutation in the index cases analyzed (9/33), of 21.2% (SD%=5.03) of the alleles (14/66) with the 35delG mutation, and of 12.5% (SD%=11.69) in the affected relatives. For each mutation, V37I and Delta (GJB6-D13S1830), a 3% prevalence (SD%=2.96) was found.The audiometric tests performed in patients with hearing impairment (n=41) showed the following results with regard to degree of loss: profound − 17 patients ; severe − 16 patients  and moderate − 8 patients . Of the five patients who were homozygous for 35delG, one presented a profound degree , three presented a severe degree , and one patient presented a moderate degree of hearing loss . Of the two patients who were heterozygous for 35delG, one  presented a severe and the other  a moderate degree of hearing loss. The patient who was a 35delG/V37I composite heterozygote  and the two 35delG/Delta (GJB6-D13S1830) composite heterozygotes  presented a severe degree of hearing loss. The predominant audiometric frequencies were 4000 to 8000 Hertz (Hz). All patients with the 35delG mutation presented hearing loss with prelingual onset.The phenotype/genotype correlation of the index cases and affected relatives, with the diagnosed mutation, is represented in In the present study, we found a 27.3% prevalence of the 35delG mutation in the index cases analyzed, 21.2% of alleles with the mutation, and 12.5% in the affected relatives. A prevalence of 3% was also assessed for each one of the mutations V37I and Delta (GJB6-D13S1830), found in the study. These results are concordant with previous studies reported in the literature, conducted in several populationsRecent studies found a 342 thousand base-pair (342 Kb) deletion close to the GJB6 [D(GJB6 - D13S1830)] gene, suggesting that this mutation could cause non-syndromic recessive hearing loss, either by a homozygous deletion or by digenic penetrance of the deletion in the GJB6 gene, associated with a trans mutation in the GJB2 gene in the heterozygous casesIn Brazil, a study conducted in newborns from the region of Sao Jose do Rio Preto, SPAccording to the literature, analyses of the GJB2 gene in patients with hearing impairment frequently demonstrate heterozygosis in about 10% to 42% of cases, in spite of the fact that most of the mutations are recessiveAccording to the audiometric results, in the present study the hearing loss was profound in 41.5% of patients, severe in 39.0%, and moderate in 19.5%, with a predominance of the high frequencies (4000-8000 Hz). In patients who were homozygous or heterozygous for 35delG, the moderate-severe degrees of hearing loss were predominant, a pattern that is in agreement with the literatureThe clinical evaluations of patients in this study do not suggest that environmental causes are the main factors, considering that an autosomal recessive transmission pattern was identified in cases with the 35delG mutation. Moreover, upon audiometric testing, these nine patients presented non-progressive audiometric findings, similar to those of patients described in the literatureThe 35delG mutation is easy to detect and the test is viable. However, since a great part of the patients with the 35delG mutation are homozygous, broader analyses of the GJB2 gene will be necessary in a high proportion of cases, in order to distinguish the common heterozygous carriers  from the heterozygous patients with DFNB1. These broader analyses and investigations , as performed in the present study, should be oriented by the clinical characteristics of DFNB1According to the literature, molecular tests associated to audiometric data can predict that a significant number of patients with the 35delG mutation will present a moderate-severe hearing loss and others are expected to present profound hearing loss


Anastomotic leakage (AL) after colorectal surgery is a serious complication. This study aimed to evaluate the effectiveness of the EEA™ circular stapler, a new triple-row circular stapler (TCS), relative to a conventional, double-row circular stapler (DCS).A total of 285 patients who underwent anastomosis with the double stapling technique at the Tokyo Medical University Hospital between 2017 and 2021 were included in this nonrandomized clinical trial with historical controls using a propensity score (PS) analysis. The primary endpoint was the risk of AL.P = .17). After case matching, AL occurred in 13 patients (11.6%) and 1 patient (1.8%) in the DCS and TCS groups, respectively, revealing a significant difference (P = .04). Cox models were created by applying PS to adjust for group differences via regression adjustment. Odds ratios for AL in the DCS group versus the TCS group were 0.31  in the entire cohort, 0.15 (95% CI: 0.02–0.64) in the regression adjustment cohort, and 0.14 (95% CI: 0.02–1.09) in the 1:2 PS-matched cohort.We performed a 1:2 PS matching analysis. Before case matching, AL occurred in 15 (7.4%) and 2 (2.4%) patients in the DCS and TCS groups, respectively, with no significant difference (PS analysis of clinical data suggested that the use of TCS contributes to a reduced risk of AL after colorectal anastomosis CTwith the double stapling technique. A successful anastomosis and subsequent healing depend on several factors, including the tension between the 2 connected portions of the gastrointestinal tract, a healthy blood supply to surrounding tissues, and the mechanical strength of the formed anastomosis.–6Postoperative colorectal anastomotic leakage (AL) is a devastating complication–9 The choice of surgical instruments greatly affects the safety of anastomotic operations from the viewpoint of mechanical strength and a healthy blood supply. Major advances have been made in the development of medical devices for rectal surgery, in particular, for rectal anastomosis with the double stapling technique (DST). A new circular stapler having a triple row of staples is expected to improve pressure resistance, as compared to the conventional, double-row circular stapler (DCS). However, the effectiveness of the new triple-row circular stapler (TCS) in reducing the risk of AL has not been fully examined or demonstrated.The use of circular stapling devices to facilitate colorectal anastomosis was first described in the 1970s. This approach is now a standard practice and has consistently demonstrated equivalent safety and efficacy to hand-sewn anastomosis, with added advantages of shorter anastomotic time and greater reproducibility.The present study aimed to compare the clinical effectiveness of TCS  and DCS using a propensity score analysis.Figure 1. This study was approved by the institutional review board of the Tokyo Medical University Hospital.A total of 285 patients from our medical records who underwent colorectal surgery with left-sided circular stapled colorectal anastomosis above 5 cm from the anal verge with the DST at Tokyo Medical University Hospital between 2017 and 2021 were included in this retrospective study using a propensity score analysis. Patients aged ≥20 years who underwent colorectal anastomosis after left colectomy, sigmoidectomy, or anterior rectal resection for benign or malignant pathology were included, and those with anastomosis <5 cm from the anal verge (they did not have DST anastomosis) or who had preoperative therapy were excluded. Patients were divided into 2 groups according to the device used for the DST (DCS and TCS groups). A conventional DCS was used until December 2020. A novel TCS device  was introduced in Japan in January 2021, and since then, our hospital has preferentially used this device. The flowchart of this study is shown in No changes were made during the study period in perioperative care or surgical principles. The same group of dedicated colorectal surgeons performed all surgeries.The anal side of the colorectum was incised with a linear stapler. The anvil of the circular stapler was secured in place, and end-to-end anastomosis was performed with the DST. The rod of the circular stapler was inserted from the opposite side of the linear staple line, piercing the rectal stump near the linear staple line. When the anvil and rod were combined, we waited 20 seconds before firing. After firing, the stapler was held for 10 seconds and then released. After anastomosis, we usually perform the observation of the anastomosis with the colonoscopy. as a defect of the intestinal wall at the anastomotic site leading to a communication between the intra- and extraluminal compartments or as an abscess adjacent to the anastomosis. Computed tomography scanning with rectal contrast was performed in patients with suspected AL in the absence of unquestionable clinical signs of peritonitis, which would require urgent surgery. The following computed tomography findings were considered suggestive of anastomotic failure: contrast leakage from inside the bowel to the pelvis and/or abdominal cavity, and abscess and/or perianastomotic collection associated with or without localized pneumoperitoneum.The incidence of AL within 30 days of surgery was recorded. AL was diagnosed according to the International Study Group of Rectal Cancer definitionThe primary outcome was the risk of AL by type of circular stapler used. Descriptive statistics were obtained for all variables. In the case of quantitative variables, normality was assessed using the Shapiro–Wilk test. Data were presented as median and range.P value of <.05.Absolute values and frequencies were calculated for qualitative variables. Then, possible relationships between quantitative and objective variables were investigated using parametric or nonparametric tests depending on variable distribution. Relationships among qualitative variables were tested using the chi-square test. Propensity score analysis was performed to adjust for heterogeneity between the DCS and TCS groups. Multivariate logistic regression was used to generate a propensity score predicting condition by a device (DCS or TCS). The following 11 covariates were included: age, sex, body mass index, American Society of Anesthesiologists (ASA) score, preoperative hemoglobin, surgical procedure , malignant disease (yes or no), diverting stoma (yes or no), operative time, anastomotic level from the anal verge (middle rectum (5–10 cm) and upper rectum (10–15 cm)), and postoperative complications (Clavien-Dindo classification >2). Each patient was assigned an estimated propensity score, which represented the patient’s predicted probability of the device selected. We specified matching IDs based on propensity scores and divided patients into 2 groups, pairing patients according to similarities in their characteristics. Then, 1:2 propensity score matching was performed. Each patient in the DCS group was matched to a patient in the TCS group who had the closest propensity score on the logit scale with a caliper of 0.2. Propensity scores were also used for regression adjustment, in which the treatment effect was estimated by adjusting for the impact of background covariates in a regression model. All statistical analyses were performed using SPSS software  and EZR , a graphical user interface for R . The level for statistical significance was set at a Table 1. The AL rate in the entire cohort was 6.0%. Patient characteristics of 2 groups are shown in Table 2. In the entire cohort, patients in the DCS group were significantly older compared to those in the TCS group (P = .01), and significantly fewer patients in the DCS group had a low ASA score compared to the TCS group (P < .001). No significant differences were observed in other covariates and AL rates between the 2 groups.Baseline characteristics of the entire cohort are summarized in Figure 2. Both DCS and TCS groups  showed a well-matched distribution with respect to patient and clinical characteristics in the adjusted analysis after case matching (Table 2). There were no significant differences in covariates other than ASA score between the 2 groups.A 1:2 propensity score–matched analysis was performed according to propensity scores to adjust for heterogeneity in the DCS and TCS groups, with 11 covariates. Distributions of propensity scores before and after case matching are shown in Table 1). Before case matching, 15 patients (7.4%) in the DCS group and 2 patients (2.4%) in the TCS group (P = .17) had AL, with no significant difference between the 2 groups (Table 2). After case matching, 13 patients (11.6%) in the DCS group and 1 patient (1.8%) in the TCS group (P = .04) had AL, with the former showing a significantly higher proportion (Table 2). Among patients who had AL after case matching, there were 6 patients in the DCS group (46%) and no patients in the TCS group (0%) who had diverting stoma.There are no critical stapler failures with intraoperative colonoscopy. AL was observed in a total of 17 patients .Regression models were created by applying propensity scores to adjust for group differences via regression adjustment. Odds ratios for AL in the DCS group versus the TCS group were 0.31  in the entire cohort, 0.15 (95% CI: 0.02–0.64) in the adjusted cohort, and 0.14 (95% CI: 0.02–1.09) in the 1:2 propensity score-matched cohort  compared to the DCS group (11.6%), suggesting that TCS can markedly reduce the risk of AL. Moreover, we found a marked improvement relative to the previously reported AL rate.–30 This mechanism may explain the greater pressure resistance of TCS. Simply tightening the conventional circular stapler may further press and crush the already tightened colon wall due to purse-string sutures and may thus result in the protrusion of a portion of the colon wall. Second, compression with staples of different heights may help maintain blood flow to the formed anastomosis.The number of staples in TCS is increased by 50% compared to DCS, with no change in outer diameter. In addition, TCS has 3 rows of staples having different heights. These features may contribute to the reduced rate of AL for the following reasons. First, compared to DCS, which has a double row of staples of the same height, TCS allows for gradual compression from the inside to the outside of the lumen, gradually releasing pressure outward, thereby preventing severe compression damage.This study has some limitations. First, we used a single-center nonrandomized clinical trial with historical controls in design. This study design introduces selection bias. In this study, the differences in background factors between the 2 groups were addressed to the extent possible using the statistical method of propensity score matching to minimize selection bias. However, due to selection bias, our results may overestimate the effect of TCS on reducing the risk of colorectal AL, compared to DCS. Second, patients who were operated on during different periods were included in this study . However, during the 5-year study period, all operations were performed by the same surgical team, including experts in colorectal surgery. A prospective study will be needed to further clarify the effectiveness of TCS.In conclusion, the present propensity score analysis suggests that the use of TCS contributes to a reduction in AL risk after colorectal anastomosis with the DST.J.M. wrote the manuscript. T.T., K.K., H.K., M.E., and T.I. acquired data for the study. All authors reviewed the manuscript. N.F., K.K., Y.N., and A.T. drafted the manuscript or revised it critically for important intellectual content.


The In2O3/InGdO TFT consisted of a highly conductive In2O3 film as the primary transmission layer and a subconductive InGdO film as the buffer layer, and exhibited excellent electrical performance. Furthermore, by altering the Gd dopant concentration, we obtained an optimal In2O3/InGdO TFT with a higher saturation mobility (µ) of 4.34 cm2V−1s−1, a near-zero threshold voltage (Vth), a small off-state current (Ioff) of on/Ioff) of 2O3/InGdO TFTs enable long-term air stability while retaining a high-mobility optimal switching property. This study suggests that the role of a high-performance In2O3/InGdO heterojunction channel layer fabricated by the solution process in the TFT is underlined, which further explores a broad pathway for the development of high-performance, low-cost, and large-area oxide electronics.The use of the semiconductor heterojunction channel layer has been explored as a method for improving the performance of metal oxide thin-film transistors (TFTs). The excellent electrical performance and stability of heterojunction TFTs is easy for vacuum-based techniques, but difficult for the solution process. Here, we fabricated In At nO, etc. ,5,6,7,8.sability , and hignologies ,11. To onologies . Choosinnologies , which inologies . Nowadaynologies ,15,16,17nologies ,17,18,19nologies . This issed TFTs . Neverthsed TFTs . Recentlsed TFTs , VZTO/ZTsed TFTs , InZnO/Ased TFTs , In2O3/Z[2O3/ZnO , In2O3/I2O3/IGZO , ZnO/In2GZO [2O3 , and MgZGZO [2O3 . These stability ,26,27,28tability ,30. Thestability ,32. Exce2O3 TFTs by the solution process. Then, the In2O3/InGdO TFTs were constructed and contrasted to single-layer In2O3 or InGdO TFTs to validate the vantages of the bilayer structure. Finally, we optimized the electrical performance of the In2O3/InGdO TFTs by varying the Gd doping concentration and reported a comprehensive high performance TFT with a higher mobility of 4.34 cm2V−1s−1, a near-zero Vth, a small Ioff of on/Ioff of In this paper, we investigated the impact of Gd doping on the electrical performance of the In2O3 precursor solution was prepared by dissolving 99.99% purity Indium nitrate hydrate (In(NO3)3.xH2O) in 2-methoxyethanol to yield a 0.05 M solution. InGdO precursor solution with different Gd doping concentrations was separately prepared by dissolving 99.99% purity In(NO3)3.xH2O in 2-methoxyethanol with moderate gadolinium nitrate hexahydrate (Gd(NO3)3.6H2O) to yield a 0.05 M solution. All reagents were purchased from Aladdin  and used without further purification. Precursor solutions were stirred overnight at room temperature, and then filtered through a 0.2 μm PTFE membrane filter before spin coating.The In2 (300 nm) as the gate insulator. Before the active layer deposition, the substrate was ultrasonically cleaned in acetone, ethanol and deionized water, sequentially, for 10 min each, and dried it using high purity N2. To improve the chemical compatibility between the interface of the SiO2 and the semiconductor layer, the surface of the cleaned substrate was hydrophilized for 10 min with O2 plasma. For the In2O3/InGdO TFTs, the In2O3 precursor solution was filtered, spin-coated onto the SiO2 at 3000 rpm for 30 s, and then annealed on a hotplate at 300 °C for 20 min. The thicknesses of the In2O3 layer were controlled by repeating the procedure. Subsequently, the InGdO precursor solution was immediately spin-coated onto the In2O3 film to avoid contamination and annealed under the same conditions. The In2O3 and InGdO films have a similar thickness of about 10 nm measured by surface profilometer. Finally, the 80 nm Al source and drain electrodes were thermally evaporated onto the semiconductor layer through a shadow mask that has a channel width (W) and length (L) of 600 and 100 µm, respectively. Similarly, the single-layer In2O3 or InGdO TFTs were fabricated for comparison. The schematics cross-sectional view of the single-layer In2O3 or InGdO and the In2O3/InGdO heterojunction TFTs are shown in The TFTs were fabricated on n-type Si substrates with thermally grown SiO2O3, InGdO and In2O3/InGdO films annealed at the identical temperature were measured by XRD, as shown in 2O3 film. On the other hand, the peak intensity of the InGdO film was significantly diminished compared with that of the In2O3 film. It implies that Gd ions may lead to the degradation of the In2O3 crystallinity to a certain extent, which is consistent with the results of previous studies regarding the disappearance of the peak of InGdO films via Gd doping [2O3 and In2O3/InGdO films do not change significantly, which demonstrates that the thinness of the InGdO film cannot affect the underlying In2O3 film. The surface morphology of In2O3, InGdO, and In2O3/InGdO films at 300 °C were checked by AFM and shown in 2O3 films over a scan area of 10 μm × 10 μm was 0.598 nm. However, the InGdO film was featureless and smooth, and its RMS roughness was 0.334 nm less than that of In2O3 film. Presumably, the Gd ions in the InGdO film mitigated the degree of random crystallization, leading to the reduced RMS roughness value, which is consistent with the previous report. Additionally, the result was also confirmed from the XRD analysis, as mentioned above. Interestingly, the surface roughness of the In2O3/InGdO film was an equalization between the In2O3 and InGdO films, and its RMS roughness value was 0.495 nm, suggesting that the InGdO capping layer can alleviate In2O3 film roughening during the deposition annealing process at 300 °C.d doping . However2O3 or InGdO single-layer and In2O3/InGdO heterojunction channel layer are shown in DS) from 0 to 30 V at different constant gate voltages (VGS) varied from 0 to 30 V in a step of 6 V. The transfer characteristics of TFTs were measured at a VDS of 10 V and a VGS swing from −20~+50 V, whereas the source electrodes were fixed to 0 V. By a linear fit plot of the square root of the IDS versus VGS, the mobility is obtained from the equation:i is the capacitance per unit area of the SiO2 layer, and Vth, VGS, W, and L denote the threshold voltage, the gate voltage, the channel width, and channel length, respectively. The Vth can be extracted by linear extrapolation of the ID1/2-VGS at saturation regions. To further investigate the electrical properties of the TFTs, the subthreshold swing (SS) values are noticed and directly reflect the switching speed of the TFT devices. SS is directly related to the trap states at the interface of the dielectric layer to the metal oxide; it can be derived as follows:The representative output and transfer curves of TFTs with In2O3 TFT exhibited a mobility of 9.34 cm2V−1s−1, Vth of −7.94 V, Ioff of 2.89 × 10−6, and Ion/Ioff of 2.65 × 102. As speculated, the InGdO TFT showed a significantly decreased Ioff of 1.79 × 10−11A and a superior Ion/Ioff of 1.30 × 107, which are desirable for practical applications. However, the on-state current (Ion) was significantly reduced in the InGdO TFTs. Simultaneously, the InGdO TFT achieved a lower mobility of 1.37 cm2V−1s−1 and a positive offset Vth, in comparison to In2O3 TFT. This is attributed to the formation of strong oxygen bonds between Gd ions and oxygen, which reduces mobility because the lack of oxygen vacancies prevents prefilling of trap states [th value for the InGdO TFT increased to approximately 5.89 V compared with that of the control In2O3 TFT. Additionally, the forward shift in Vth is caused by the need for a larger gate voltage to induce more carriers to prefill traps [th indicates a lower mobility in the channels [2O3 TFT with a very unsatisfactory SS. The tremendous enhancement in SS illustrates that the concentration of the traps at the SiO2/InGdO interface is greatly diminished for the InGdO TFT [2O3/InGdO TFT presented a noteworthy mobility, Vth, improved Ion/Ioff, and acceptable Ioff and SS, as shown in GS GS is more than the Vth, the dominant current path formed by the electron aggregation between the insulating layer and the In2O3 layer creates a high-concentration carrier channel. The InGdO thin film plays a vital position as the buffer layer; it can drastically decrease the Ioff of the In2O3/InGdO TFT. The In2O3 film provides enough current carriers; therefore, it enhances the Ion/Ioff of the device. It should be emphasized that the mobility value of the In2O3/InGdO TFT reaches up to 5.38 cm2V−1s−1, surpassing that of the InGdO TFT, which is accredited to the In2O3 layer with extremely high mobility. The Ion/Ioff, SS, Vth and Ioff values of In2O3/InGdO TFT were also improved to 9.01 3, 7.68 V/dec, −3.77 V and 7.86 × 10−8 A, respectively, compared with those of the In2O3 TFT. The superior performance of the In2O3/InGdO TFTs strongly suggests that the In-based TFTs were effectively optimized without significantly sacrificing the carrier mobility by adopting the In2O3/InGdO heterojunction structure. It can be inferred that the InGdO capping layer on the In2O3 film further reduced the trap states through the controlled crystallization during the deposition annealing process, as mentioned in the XRD and AFM analyses, and is also partially responsible for the superior performance of the In2O3/InGdO heterojunction device.Based on p states ,33. The channels . Concurr2O3, InGdO, and In2O3/InGdO TFTs under positive bias stability (PBS) were investigated to analyze the electrical stability of the devices, respectively, as illustrated in th. The PBS of the TFTs were conducted with a consecutive 6-time sweep under VGS swept in the range of −20 to +50 V and VDS fixed at high positive bias of +10 V for a duration of 1 h at room temperature. From 2O3 TFT exhibits a high ΔVth of 1.71 V, which is due to the numerous oxygen vacancies at the bulk region or the massive trap states at the SiO2/In2O3 interface [th instability. When the PBS test was applied in the atmosphere, excess electrons accumulated in the channel layer and were captured by the surrounding oxygen molecules, which exhausts electron carriers and leads to the Vth positive shift [2O3 or the strong oxygen binders Gd prevent the desorption of oxygen from the atmosphere [2O3/InGdO TFT was shown in th value of 1.26 V, which is essentially due to the total amount of oxygen vacancies obviously decreased from the In2O3 to the In2O3/InGdO; it is partially responsible for the superior PBT stability of the In2O3/InGdO TFTs. Moreover, it may also be one of the reasons that the strong oxygen binder Gd prevents the desorption of oxygen in the air.The transfer curves for Innterface ,35. In ave shift . The InGmosphere . Additio2O3/InGdO TFTs with different Gd doping concentrations. The morphology of the In2O3/InGdO thin films was analyzed for understanding the dependence of the properties of the In2O3/InGdO TFTs on the Gd doping concentration, as shown in 2O3/InGdO films were 0.481 nm, 0.459 nm, 0.422 nm and 0.401 nm, respectively. There was no obvious difference in RMS roughness levels of less than 1 nm, illustrating that the samples are smooth and continuous, which is conducive to strengthening their adhesion and forming a good Ohmic contact between the channel layers and Al source/drain electrodes. Next, the In2O3/InGdO TFTs with Gd concentrations increasing from 3% to 5%, 7%, and 9% were fabricated and measured, as indicated in 2V−1s−1, and the Vth gradually rises from −4.61 to −3.77, 0.97, and 2.40 V. Because the oxygen vacancy in the channel layer was suppressed by the Gd ions, less oxygen vacancy which remains in the channel layer ultimately induces a lower conductive In2O3/InGdO channel. The improvement of the Ion/Ioff from 1.84 × 103 to 9.01 × 103, 3.18 × 105, and 3.71 × 105 is owing to the low Ioff derived from the higher Gd doping concentration in the InGdO layer, and the guaranteed Ion originated from the In2O3 layer thus leads to the increase in Ion/Ioff. Additionally, the SS varies from 8.10 to 7.68, 4.54, and then to 3.18 V/dec, which proves that the SS of devices can be effectively improved by modulating the buffer layer via changing the concentration of Gd doping ions in the heterojunction channel TFTs.To further optimize the performance of the heterojuction TFTs, we fabricated and investigated In2O3/InGdO TFTs, as shown in th value decreased slightly when the content of Gd ions increased from 3% to 5%, which is due to fewer oxygen vacancies existing in the channel layer because of the introduction of more Gd ions in higher concentrations of doping samples. The ΔVth value increased again when the content of Gd ions increased from 7% to 9%, and the devices showed a larger ΔVth with stress time. Generally, SS values reflect ΔVth, which is related to the fast bulk traps and the interface trap density between the dielectric and the semiconductor [th, SS and ΔVth are shown in 2O3/InGdO TFTs demonstrate optimum electrical performance and stability at 7% Gd content, which has been verified by several of the experiments.The evolution of the transfer curves of the TFTs under the PBS was studied to further investigate the influence of the Gd doping concentration on the stability of Inonductor ,37. Thusonductor . The var2O3/InGdO TFTs after three months are shown in th. Aging tests have proven that In2O3/InGdO TFTs can accomplish long-term air stability while maintaining optimal switching property at high mobility. The most intriguing part is that the mobility values of the TFTs increase from 2.41 to 2.42, 3.12, and 3.36 cm2V−1s−1, which is contrary to the increasing doping concentrations from 3% to 5%, 7%, and 9%. Additionally, the Vth values for In2O3/InGdO TFTs changed, with 3%, 5%, 7% and 9% Gd concentrations gradually increased from −4.86 to 0.94, 7.46 and 13.19 V, as shown in th. In addition, we found that the mobility values of the TFTs change from 6.22 to 2.41 cm2V−1s−1 for 3% Gd-doped In2O3/InGdO TFT, from 5.38 to 2.42 cm2V−1s−1 for 5% Gd-doped In2O3/InGdO TFT, from 4.34 to 3.12 cm2V−1s−1 for 7% Gd-doped In2O3/InGdO TFTs, and from 2.54 to 3.36 cm2V−1s−1 for 9% Gd-doped In2O3/InGdO TFTs after three months. The corresponding values of the mobility change for In2O3/InGdO TFTs with 3%, 5%, 7% and 9% Gd concentrations gradually diminished from 3.81 to 2.96, 1.22 and 0.82 cm2V−1s−1; the specific trends are shown in The transfer characteristics as a function of the Gd doping concentration of In2O3/InGdO heterojunction channel layer using a solution process, and compared the electrical properties with single-layer In2O3 TFTs and InGdO TFTs. In our results, the InGdO TFT showed a higher Ion/Ioff of 1.30 × 107, a smaller SS of 1.75 V/dec, and a smaller ΔVth of 0.85 V. Unfortunately, the InGdO TFT achieved a lower mobility of 1.37 cm2V−1s−1 in comparison to the In2O3 TFT. However, the In2O3/InGdO TFT, composed of a highly conductive In2O3 film as the primary transmission layer and a subconductive InGdO film as the buffer layer, showed high electrical performance with higher mobility and higher Ion/Ioff. The results indicate that the high carrier concentration the In2O3 layer close to the insulation layer ensures the excellent mobility of In2O3/InGdO TFTs and low carrier concentration InGdO layer can reduce the Ioff of the devices, thereby increasing the Ion/Ioff of the devices. Furthermore, the PBS test results indicate that TFTs with In2O3/InGdO heterojunction channels are much more stable. In addition, to further optimize the performance of the heterojunction TFTs, the influence of the Gd doping concentration have been thoroughly studied. The results show the 7% Gd-doped In2O3/InGdO TFT has optimum characteristics, displaying a superb mobility of 4.34 cm2V−1s−1, a close-to-0 Vth of 0.97 V, an SS of 4.54 V/dec, a high Ion/Ioff of 3.18 × 105, and a small ΔVth of 1.83 V. Finally, an aging test was carried out after three months, indicating that the In2O3/InGdO TFTs enable long-term air stability while retaining an optimal switching property of high mobility. The work demonstrates that the heterojunction channel structure of solution-processed metal-oxide semiconductors is feasible for fabricating the high performance TFTs, which further explores a broad pathway for the development of high-performance, low-cost, and large-area oxide electronics.In conclusion, we fabricated TFTs with the In


Telocytes (TCs), a novel type of interstitial cells, have been found to participate in tissue protection and repair. In this study, we investigated the antioxidative effects of TCs in inflamed lungs of mice. Acute respiratory distress syndrome (ARDS) mice were used as models of inflamed lungs of mice. Gene sequencing was used to screen the differentially expressed miRNAs in TCs after lipopolysaccharide (LPS) stimulation. AntagomiR-146a-5p-pretreated TCs were first injected into mice, and antioxidant activity of TCs was estimated. TCs, RAW264.7 cells, and MLE-12 cells were collected for the detection of expressions of NOX1–4, DUOX1–2, SOD1–3, GPX1–2, CAT, Nrf2, miR-146a-5p, and miR-21a-3p after LPS stimulation. Silencing miRNAs were delivered to examine the involved signaling pathways. Oxidative stress was examined by measuring malondialdehyde (MDA) levels. We found that microRNA-146a-5p and microRNA-21a-3p were upregulated in TCs after LPS stimulation. ARDS mice that were preinfused with TCs had lower lung tissue injury scores, lung wet-dry ratios, white blood cell counts in alveolar lavage fluid and lower MDA concentrations in lung tissue. However, in antagomiR-146a-5p-pretreated ARDS mice, the infusion of TCs caused no corresponding changes. After LPS stimulation, DUOX2 and MDA concentrations were downregulated in TCs, while DUOX2 was restored by antagomiR-146a-5p in TCs. Dual-luciferase reporter assay confirmed that CREB1 was downregulated by miR-146a-5p, while DUOX2 was downregulated by CREB1, which was confirmed by treating TCs with a specific CREB1 inhibitor. This study demonstrates that LPS stimulation upregulates miR-146a-5p in TCs, which downregulates the CREB1/DUOX2 pathway, resulting in a decrease in oxidative stress in cultured TCs. TCs reduce LPS-induced oxidative stress by decreasing DUOX2 in inflamed lungs of mice. Acute respiratory distress syndrome (ARDS) is an acute clinical symptom characterized by diffuse alveolar cell damage and severe injury of the respiratory alveolo-capillary barrier followed by non-cardiogenic pulmonary edema and hypoxic dyspnea, due to widespread inflammation in the lungs –4. EnhaIn the structure of lung telocytes (TCs), a novel type of interstitial cells were described soon after their discovery in 2010 . These pin vitro experiments, TCs were co-cultured with primary human dermal microvascular endothelial cells (HDMECs) as well as mouse fibroblast cells (L929), and the results showed that TCs significantly reduced LPS-induced oxidative stressSV40 declined with the increase of LPS or TNFα concentration, similar to the responses of primary TCsSV40 may be a useful tool in the development of challenging new therapies.In our previous study, we found that exogenous TCs protect against LPS-induced pulmonary injury by reducing cytokine levelsin vitro andin vivo [MicroRNAs (miRNAs), which are small noncoding ribonucleic acid molecules, regulate target genes by inhibiting mRNA translation or promoting mRNA degradation. Moreover, miRNAs have been found in the extracellular vesicles released by TCs, bothin vivo ,32. It in vivo ,37.In the present study, we aimed to investigate the antioxidative effects of TCs in the inflamed lungs of mice, with a special focus on the underlying mechanisms of noncoding RNAs functioning on the enzymes involved in oxidative stress in TCs.LPS was purchased from Sigma . Antibodies against CREB1 (ab31387) and phosphorylated CREB1 (ab32096) were purchased from Abcam . Antibody against DUOX2 (NB110-61576ss) was purchased from NOVUS . Specific inhibitors of CREB1, KG-501 (HY-103299) and 666-15 (HY-101120) were purchased from MedChem Express , Lipofectamine RNAiMAX was purchased from Thermo Fisher Scientific . Bicinchoninic acid assay (BCA) kit, RIPA lysis buffer, and loading buffer were purchased from Beyotime .SV40SV40 cells were cultured in Dulbecco’s modified Eagle’s medium/F12  supplemented with 10% fetal calf serum . Mouse RAW264.7 and MLE-12 cells were obtained from Zhongqiao Tech . All the cells were cultured in DMEM/F12. HEK293T cells  were cultured in DMEM high glucose medium  for the Dual luciferase reporter assayMouse lung TCs were kindly provided by Dr Xiangdong Wang , and cells were transfected with SV40 large and small T antigen to constructed TCs6 cells) were freshly prepared in 100 μL PBS.Cell transfection was performed using Lipofectamine RNAiMAX (Thermo Fisher Scientific) according to the manufactory’s protocol. In brief, 50 nM antagomiR-146a-5p  was prepared in a serum-free and antibiotic-free medium. After transfection, TCs were supplied with full growth medium until confluence. For animal experiments, TCs (1×106 cells) transfected with antagomiR-146a-5p or not, were administered via the tail vein 2 h before the LPS stimulation. The animals were sacrificed, and the lungs and bronchoalveolar lavage fluid were collected 24 h after the establishment of the mouse ARDS model.Twenty-four male C57BL/6 mice  were obtained from the Shanghai SLAC Animal Company  and kept in a pathogen-free condition. The mice were housed in standard air-conditioned rooms (22–24°C) under a 12/12 h light/dark cycle, with free access to food and water. Mice were randomly divided into four groups: the control group, LPS stimulation group, TCs treatment group challenged with LPS, and miR-146a-5p-silenced TCs treatment group challenged with LPS. The mouse ARDS model was established by perfusing mice with LPS (5 mg/kg) via the trachea. TCs .g, white blood cells were counted under a microscope . The left lung was fixed in 10% formalin solution and embedded in paraffin for hematoxylin and eosin (H&E) staining. The right lung was collected to assess edema by the ratio of wet weight/dry weight (W/D) of the lung after incubation in a 60°C oven for 48 h. The lung injury was evaluated using a modified lung injury histological scoring systemTo collect white blood cells in bronchoalveolar lavage fluid, mice lungs were perfused with ice-cold phosphate-buffered saline (PBS). After centrifugation at 3000U6 and mRNAs expressions were normalized withGAPDH. Primers used in the present study were synthesized by Sangon Biotech  as listed inTotal RNA was extracted from cultured TCs using Trizol reagent . MicroRNAs were reversely transcripted with a Bulge-Loop miRNA qRT-PCR Starter kit (Ribo Biotech), and mRNAs were reversely transcripted to complementary DNA (cDNA) using a PrimeScript RT Reagent kit with gDNA Eraser (TaKaRa). Both miRNAs and mRNAs were amplified on an ABI 7500 sequence detection system . The expression levels of miR-21a-3p (Cat. MQPS0002615) and miR-146a-5p (Cat. MQPS0002462) were normalized withCultured cells were lysed in RIPA buffer containing protease inhibitors , phosphatase inhibitors (Beyotime), and phenylmethanesulfonyl fluoride . Total protein concentration was determined using the bicinchoninic acid assay kit (Beyotime). Samples (30 μg) were subject to SDS-PAGE and then transferred onto polyvinylidene fluoridewere (PVDF) membranes. Mebranes were blocked with 5% bovine serum albumin in Tris-buffered saline containing 0.1% Tween 20. The membranes were then incubated with primary antibody at 4°C for overnight. On the second day, the membranes were further incubated with an HRP-conjugated anti-rabbit IgG secondary antibody  for 1 h. Finally, target protein signals were visualized with Omni-ECL Femto Light Chemiluminescence kit  on a Fluorescent Image Detection System . The related signals were quantified using ImageJ software and normalized with GAPDH. The information of all the antibodies are shown as follows: antibodies against JAK1 (CST-3344), JAK2 (CST-3230), JAK3 (CST-8827), TYK2 (CST-14193), STAT1 (CST-14994), STAT2 (CST-72604), STAT3 (CST-30835), STAT4 (CST-2653), STAT5 (CST-94205), STAT6 (CST-5397), phosphorylated JAK1 (CST-74129), phosphorylated JAK2 (CST-8082), phosphorylated JAK3 (CST-5031), phosphorylated TYK2 (CST-68790), phosphorylated STAT1 (CST-7649), phosphorylated STAT2 (CST-4441), phosphorylated STAT3(Try705) (CST-9145), phosphorylated STAT3 (Ser727) (CST-9134), phosphorylated STAT5 (CST-4322), phosphorylated STAT6 (CST-9316), and GAPDH (CST-5558) were purchased from Cell Signaling Technology.The PHY-881 reporter vector  was used to construct the luciferase reporter vector plasmids PHY-881-WT-CREB1-3′-UTR and PHY-881-Mut-CREB1-3′-UTR. The plasmids CREB1-WT or CREB1-Mut were co-transfected with miR-146a-5p mimic  or negative control  into human embryonic kidney (HEK293T) cells using Lipofectamine 2000 reagent (Thermo Fisher Scientific). A dual-luciferase reporter assay system  was used to assess luciferase activity after 24 h of transfection. Relative firefly luciferase activity was normalized to renilla luciferase activity.Malondialdehyde levels in TCs and culture medium were measured using malondialdehyde (MDA) assay kit  according to the manufacturer’s instructions. Briefly, culture medium or cell lysates were incubated in MDA working solution at boiling temperature for 15 min. The MDA concentration was determined by measuring the absorbances at 532 nm, and normalized with protein concentrations in culture medium or cell lysates, respectively.P<0.05 was considered statistically significant.Data were presented as the mean±standard deviation. The data were analyzed using the GraphPad Prism software . One-way analysis of variance (ANOVA) was used for comparison.In our previous studies, microarray sequencing was performed to identify differential exppressions of genensMDA is the product of lipid oxidative stress, and it is correlated with the severity of oxidative stress. The MDA level was found to be increased in the lung tissue of ARDS mice, but decreased in the TCs-treated group mice, and increased again when infused with TCs pretreated with antagomiR-146a-5p A. MDA leDUOX2 is the candidate gene distinguishing TCs from RAW264.7 and MLE-12, DUOX2 protein expression was measured in the three cells stimulated with LPS. DUOX2 protein level was significantly increased in RAW264.7 and MLE-12 cells but significantly reduced in TCs after treatment with LPS , NOX2, dual oxidase 1 (DUOX1), and DUOX2, but significantly decreased mRNA expressions of antioxidant enzymes superoxide dismutase 3 (SOD3), glutathione peroxidase 1 (GPX1), GPX2, and catalase (CAT) A. In MLEith LPS C. Silencith LPS D.FigurCREB1 promoter activity in HEK293T cells, but not in HEK293T cells with mutatedCREB1  were significantly increased A, but diThe present study reports that DUOX2 protein plays a vital role in the protective effect of TCs in the mouse ARDS model. After LPS stimulation, DUOX2 protein expression is decreased in mouse TCs, which is regulated by transcription factor CREB1. Enhanced expression of miR-146a-5p inhibits CREB1 transcription, resulting in reduced expression of DUOX2 protein as well as a decrease in oxidative stress.The enzyme DUOX2, a member of the nicotinamide adenine dinucleotide phosphate (NADPH) family, has a peroxidase homology domain and a gp91phox domainDUOX2 protein is regulated at a transcriptional level. In cultured human pancreatic cells, interferon-gamma activates STAT1, with increased phosphorylation on tyrosine 701 residue, leading to the upregulation of DUOX2 protein ,42. In MicroRNAs (miRNAs), small non-coding ribonucleic acid molecules, regulate target genes by inhibiting mRNA translation or promoting mRNA degradation ,44. It In conclusion, miR-146a-5p in TCs downregulates the expression of DUOX2 via the CREB1 pathway and administration of TCs protects the function of the lung undergoing oxidative stress damage .Figure


Genome-wide association studies (GWAS) was used to identify the candidate and potential SNPs for three beak colors in ducks . The results showed that 2753 significant SNPs were associated with black beaks, 7462 with yellow, and 17 potential SNPs with spotted beaks. Based on SNP annotation, MITF, EDNRB2, members of the POU family, and the SLC superfamily were the candidate genes regulating pigmentation. Meanwhile, isoforms MITF-M and EDNRB2 were significantly different between black and yellow beaks. MITF and EDNRB2 likely play a synergistic role in the regulation of melanin synthesis, and their mutations contribute to phenotypic differences in beak melanin deposition among individuals. This study provides new insights into genetic factors that may influence the diversity of beak color.Beak color diversity is a broadly occurring phenomenon in birds. Here, we used ducks to identify candidate genes for yellow, black, and spotted beaks. For this, an F Beak pigmentation is a common phenomenon observed in most birds. The color of feathers, coats, and skin is primarily determined by melanocytes, which are involved in the synthesis of melanin and play an important role in cosmetic change, heat regulation, camouflage, and protection against UV radiation from sun exposure ,2,3. In TYR, IRF4, TYRP1, OCA2, SLC45A2, MC1R, and KITLG genes are significantly associated with human skin color [MC1R [AIM1 or MATP) encodes a transporter that mediates melanin synthesis and is expressed in a high percentage of melanoma cell lines [SLC45A2 mutations have been reported to lead to OCA4, and polymorphisms of this gene are significantly associated with human skin, hair, and eye pigmentation [SLC24A5, SLC45A2, TYR, and APBA2/OCA2) [With rapid development in genetics and genomics, researchers have gradually realized that human skin color diversity is due to the natural positive selection of those genes that affect human pigmentation, especially in melanosome biogenesis or melanin biosynthetic pathways ,12,13,14in color ,16,17. Mor [MC1R . Furtherll lines ,20. Seveentation ,22,23. Sentation . HoweverA2/OCA2) . 2 cross between the Cherry Valley Duck and Runzhou White Crested Duck, we performed a genome-wide association study (GWAS) with 275 birds to gain insight into the effects of genetic factors on beak pigmentation. These studies provided insight into the molecular regulatory mechanisms and genetic improvement of melanin deposition in duck beaks. Ducks, the second-largest poultry species in the world, mostly have yellow and black beaks. Occasionally, spotting occurs, which directly affects carcass sales. However, genetic factors that lead to the appearance of spot color remain unclear. Based on an FAll experiments on ducks were performed in accordance with the Regulations on the Administration of Experimental Animals issued by the Ministry of Science and Technology  in 1988 (last modified in 2001). The experimental protocols were approved by the Animal Care and Use Committee of the Yangzhou University (YZUDWSY2017-11-07). All efforts were made to minimize animal discomfort and suffering.2 resource population, which crosses the Chinese Crested duck (CC duck) and Cherry Valley duck (CV duck), was obtained from the Laboratory of Poultry Genetic Resources Evaluation and Germplasm Utilization at Yangzhou University. The ducks were raised in stair-step cages under the recommended environmental and nutritional conditions at the conservation farm of Ecolovo Group, China. The CC duck is a Chinese indigenous breed that has a black shank and beak, while the CV duck is a commercial breed that has a yellow shank and beak and white plumage. In the F1 generation, 30 CC ducks and six CV ducks were randomly selected and divided into six families to interbreed. To generate F2 progeny, 30 males and 150 unrelated female ducks were used as hybrids. A total of 275 ducks were used in the next experiment. To identify candidate genes associated with beak color, we classified beak color as yellow, spotted, or black. The R/ggcor package was used to calculate the correlation between beak color and sex.The FBlood samples were used to collect high-quality DNA at 42 days of age. Genomic DNA (gDNA) was extracted from blood samples by using the DNA extraction kit method (QIAampR DNA Blood Mini Kit), following the manufacturer’s protocol. Two paired-end sequencing libraries with insert sizes of 350 bp were constructed according to the Illumina protocol . All libraries were sequenced using the Illumina NovaSeq platform.Raw reads were filtered using the NGS QC Toolkit (version 2.3) with default parameters . The cleThe population structure was assessed with multidimensional scaling (MDS) using PLINK 1.9 software . Indepeny is a vector of beak colors, X is the incidence matrix for a random additive effect, a is the column vector of random additive effects, Z is the genotype value of the candidate SNP, b is the regression coefficient of the candidate SNP, and e is the random residual. The phenotypic variance–covariance matrix is var(y) = Var(a) + var(e) = a2K σ + e2I σ, where K is the IBS kinship matrix, I is the identity matrix, a2σ is the additive variance, and e2σ is the variance of random residuals. The regional Manhattan plot and LD heatmap were obtained using LDBlockShow software. The GWAS analysis of beak color used the linear mixed model in the Effective Mixed Model Association eXpedited (EMMAX) software . EMMAX mBased on the LD attenuation distance calculated using PopLDdecay , annotatTo identify the candidate genes for beak color, we first focused on the correlation between beak color and sex to determine whether there was a correlation between these two traits. The results showed that there was no correlation between black, yellow, or spotted beak color and sex a. We alsEMMAX software was used to conduct the genome-wide association analysis in the present study. The Q–Q plot illustrated that the model used for the GWAS analysis was reasonable. The lambdas (inflation factor (λ)) of the three different color beaks were 0.98 (black beak), 1.05 (spotted beak), and 0.99 (yellow beak), and the points at the upper right corner of the Q–Q plots were the significant markers associated with the traits under study . Thus, pp-value , and 1916 extremely significant SNPs were identified using the threshold of significant p-value , most of which were located on chromosome 14 (ALP 14) (2708 SNPs) and ALP 11 (45 SNP)  , top. p-value , and 5878 extremely significant SNPs were identified using the threshold of significant p-value , most of which were located in ALP11 . However, we identified 17 potential SNPs associated with spotted beaks using the threshold of significant p-value   , bottom.× 10−08) . In addiMITF, EDNRB2, SLC25A43, SLC25A5, SLC25A14, SPRY3, POU4F3, etc.  family were significantly associated with yellow beaks. Although we did not identify a significantly associated signal in the spotted beak, we found two candidate signal loci on chromosomes 11 and 14. By annotating the candidate signals, we found that MITF and EDNRB2, two key genes responsible for melanin synthesis, were enriched. Melanin is a substance in the body that is responsible for hair, eye, and skin pigmentation . MelaninMITF has been shown to affect pigmentation in cattle [MITF belongs to the basic helix–loop–helix–leucine zipper (bHLHZip) family of proteins. Studies have shown that it regulates melanogenesis by binding to the highly conserved M-box (GTCATGTGCT) and E-box (TCATGTG) motifs upstream of the tyrosinase promoter, thereby strongly stimulating and promoting the activity of the tyrosinase promoter. Tyrosinase expression promotes melanin production [MITF, MC1R, EDNRB2, the PMEL family, TYR, TYRP1, and TYRP2, affect melanin syntheses. Our findings showed similar results, including a significant association of MITF. n cattle ,42,43,44n cattle ,46,47, hn cattle ,49,50,51n cattle ,53, human cattle ,55, and n cattle . MITF beoduction ,58,59,60EDNRB is a gene expressed in melanocytes that are derived from the neural crest, and for this reason, EDNRB is particularly mentioned. EDNRB2 is a homolog of EDNRB, which belongs to the endothelin receptor (EDNR) gene family and has been lost in mammalian lineages. EDNRB signaling is required for melanocyte development [EDNRB2 regulated melanin pigmentation in ducks. In addition, our results showed that the spotted beak was mainly coregulated by MITF and EDNRB2. However, the mechanism by which MITF and EDNRB2 are coregulated requires further investigation.elopment ,62. Losselopment ,64, and elopment ,66. A siMITF [POU4F1, POU3F3B, POU1F1, and POU2F1 were identified. Therefore, we speculated that the yellow beak is coregulated by the POU family and MITF, but the specific mechanism requires further experimental verification. This study provided new clues for understanding the genetic factors that may influence the diversity of beak color, but further experimental studies are needed to strengthen this hypothesis.Our results suggested that the POU family and SLC superfamily are significantly associated with yellow beaks. Yellow skin, beaks, and feet in most birds are caused by carotene deposits. The results showed that most members of the POU family, which share the typical POU domain structure , were siMITF , and POUMITF and EDNRB2 were the candidate genes associated with beak melanosis. We speculated that beak pigmentation may be coregulated by the POU family, MITF, and EDNRB2. However, the specific mechanisms require further experimental verification.This study identified candidate genes closely related to duck beak color.


Objective. Systemic administration of dieckol reportedly ameliorates acute hearing loss. In this study, dieckol was delivered to the inner ear by the intratympanic route. The functional and anatomic effects and safety of dieckol were assessed using the rat ototoxicity model. Materials and methods. Dieckol in a high-molecular-weight hyaluronic acid vehicle (dieckol+vehicle group) or vehicle without dieckol (vehicle-only group) were randomly delivered into 12 ears intratympanically. Ototoxic hearing loss was induced by intravenous administration of cisplatin, gentamicin, and furosemide. The hearing threshold and surviving outer hair cells (OHC) were enumerated. Biocompatibility was assessed by serial endoscopy of the tympanic membrane (TM), and the histology of the TM and the base of bulla (BB) mucosa was quantitatively assessed. Results. The hearing threshold was significantly better (difference of 20 dB SPL) in the dieckol+vehicle group than in the vehicle-only group. The number of surviving OHCs was significantly greater in the dieckol+vehicle group than in the vehicle-only group. There were no signs of inflammation or infection in the ear. The thickness of the TM and the BB mucosa did not differ between the two groups. Conclusion. Intratympanic local delivery of dieckol may be a safe and effective method to prevent ototoxic hearing loss. More than 80% of children with cancer will be cured, but sequelae (such as hearing loss) after chemotherapy can degrade their quality of life . A methoThe main mechanism of ototoxic hearing loss is excessive oxidative stress. Production of reactive oxidative species (ROS) and nitric oxide (NO) induce hair cell loss . AntioxiEcklonia cava is an edible brown alga found mainly in South Korea, Japan, and China [E. cava (PPEE) contains several phytochemicals, including dieckol, eckol, and phlorofucofuroeckol-A, referred to as eckols [E. cava extract was approved by the U.S. Food and Drug Administration (FDA) in 2008 (FDA-1995-S-0039-0176). Dieckol promotes recovery of the inner ear after ototoxic insult [Seaweeds with a variety of phytochemical and pharmacological properties are used as substitutes for drugs . Ecklonind China . The purs eckols . Among ts eckols ,21,22,23c insult  and acutc insult . The resWe evaluated the effect of dieckol on ototoxic hearing loss. To avoid systemic effects and enhance the local treatment effect, we delivered the drug via the IT route. Our hypothesis was that the antioxidant effect of IT-delivered dieckol would prevent ototoxic hearing loss and improve hearing outcomes.p = 0.937).The external auditory canal and tympanic membrane were observed by TM endoscopy. None of the 12 ears were inflamed in either group . In bothp = 0.015 at PHD 1, p < 0.015 at PHD 4, p = 0.04 at PHD 8, p = 0.093 at PHD 12, p < 0.009 at PHD 21, p < 0.004 at PHD 30, and p < 0.009 at PHD 45) at 32 kHz. A significant difference at 8 kHz was observed on PHD 1 (p = 0.015). When the hearing at 32 kHz was compared between the control group and vehicle-only group, it was almost identical, with no statistical difference. The degree of hearing deterioration was less in the dieckol+vehicle group than the control group  at 32 kHz. As for the low to mid frequencies (8–16 kHz), the variability was very large, but a similar tendency was found.The hearing threshold was normal  in all rats before the experiment. At PHD 1, hearing had deteriorated in all rats . The degp = 0.002). The middle turn (16 kHz) also showed a superior treatment outcome (77.5 ± 5.7 cells/200 μm) compared to the vehicle-only group . In the apical turn (8 kHz) and the hook portion, the survival of OHCs in the dieckol+vehicle group  was superior to that of the vehicle-only group , but this difference did not reach a statistical significance , the middle turn (16 kHz), the basal turn (32 kHz), and the hook portion are shown in ificance B.p = 0.937). The mean thickness of the mucosa at the BB was 30.9 ± 12.3 μm in the dieckol+vehicle group and 27.7 ± 9.5 μm in the vehicle-only group (p = 0.394).The mean thickness of the TM was 2.0 ± 0.2 μm in the dieckol+vehicle group and 2.0 ± 0.3 μm in the vehicle-only group . Although dieckol is reportedly effective for noise-induced hearing loss , this isThe mechanism by which dieckol prevents ototoxic hearing loss is hypothetical—its antioxidant activity likely plays a major role. The main mechanism of ototoxicity of aminoglycosides is the production and accumulation of ROS ,27, leadThe advantages of IT drug delivery include (1) reduced systemic metabolism, (2) specific drug delivery to the inner ear, and (3) a higher concentration of drug . BecauseThe downside of IT drug delivery is middle ear infection. Various biocompatible materials and drugs that are safe in other organs can cause inflammation and/or infection in the structurally vulnerable TM ,48. TherIT delivery of dieckol resulted in a better hearing outcome at the high frequency (32 kHz) than at the middle and low frequencies (16 and 8 kHz). This is likely to be because of the diffusion property of the round/oval window . DieckolThis study had several limitations. A larger number of animals would have increased the statistical power. However, we identified a marked difference between the two groups in hearing threshold and HC count. The mean difference between the dieckol+vehicle group and vehicle-only group in PHD45 was 22.5 dB. The standard deviation of the two groups was 8.01 and 12.11, respectively. When the alpha error was set as 0.05 and the beta error was set as 0.2, the required sample size turned out to be 4 samples for each group. Accordingly, the statistical power seems to be sufficient despite the small sample size, thanks to the large mean difference and small standard deviation. This study is a proof-of-concept pilot study that points out the possibility of delivering dieckol by the IT route as a novel approach that has never been reported in the literature. Further studies should be repeated with a larger sample size for the drug to be considered for any human trial. We did not evaluate the mechanism by which dieckol promoted hearing recovery. Instead, we focused on expanding the indication and mode of administration because the mechanism has been established . We aimen = 6) and vehicle-only group (n = 6), and control group (n = 10) for comparison. As for the control group, no treatment was performed after induction of ototoxic hearing loss. The age-matched control group was recruited from our former study that was performed with the same experimental settings [All animal experiments were approved by the Animal Research Committee of Seoul National University Hospital, and animal care was supported and supervised by the institution (IACUC16-0243-01A02). Twenty-two ears of male Sprague-Dawley rats  were used. The rats were anesthetized with zoletil and xylazine. Dieckol was delivered to the inner ear via IT injection. Under a surgical microscope, one ear of each rat was randomly selected for administration of dieckol in a hyaluronic acid (HA)-based vehicle. The other ear (control ear) was administered HA vehicle only. The ears were divided into the dieckol+vehicle group , gentamicin  and furosemide  for two consecutive days induced ototoxic hearing loss. Cisplatin and gentamicin solutions were slowly (0.2 mL/2 min) injected through the vein on the lateral side of the tail using an Angiocath Plus 24-gauge needle. Five minutes after flushing the tube with normal saline (0.3 mL), furosemide was slowly (0.2 mL/2 min) injected. The last day of ototoxic hearing loss induction was considered post-hearing loss day (PHD) 0. Therefore, PID 4 was the same as PHD 0.E. cava plant was collected off the coast of Jeju Island, South Korea. Dried E. cava powder was extracted with 70% aqueous ethanol and partitioned between water and ethyl acetate. The ethyl acetate fraction was subjected to octadecylsilyl (ODS) column chromatography followed by gel filtration in a Sephadex LH-20 column equilibrated with methanol. Final purification was accomplished by HPLC (Waters Spherisorb S10 ODS2 column (20 × 250 mm); eluent, 30% methanol; flow rate, 3.5 mL/min) to isolate dieckol (98.5 wt%). As the vehicle, 2 wt%, 5000–10,000 kDa high-molecular-weight HA was used . The dieckol+vehicle was prepared as follows: 0.2 g of dieckol was mixed with 3 mL of 10% ethanol and 0.9 mL of normal saline at 37 °C and centrifuged for 30 min. Next, 16.1 mL of high-molecular-weight HA were stirred into the solution for 24 h.Dieckol was a light brown powder and was provided by Botamedi, Inc. ; its specifications are available elsewhere . BrieflyA 2.7-mm-diameter endoscope  was connected to a smartphone  to photograph the external auditory canal and TM of the rats. We evaluated signs of inflammation, infection, swelling, redness, perforations, and other side effects. TM endoscopic measurements were performed at PID 0, 1, 5, 8, 12, 16, 25, 34, and 49.Using the Smart EP system , auditory brainstem response (ABR) thresholds were evaluated at 8, 16, and 32 kHz. The rats were anesthetized as above. Subdermal needle electrodes were inserted at the vertex (active electrode) and behind the ipsilateral ear (reference electrode) and contralateral ear (ground electrode). The speaker was aligned with the external auditory canal, and the earphone tube was inserted gently into the ear canal. Hearing thresholds were assessed by evaluating the lowest stimulus level that produced clear III/V and SN10  waves. ABR testing started at 90 dB SPL and decreased in 5 dB increments. ABRs were evaluated at PRE, PID 0, and PHD 1, 4, 8, 12, 21, 30, and 45.On the last day of the experiment (PID 49), the animals were anesthetized, and the cochleae were harvested, perfused with 4% paraformaldehyde in phosphate-buffered solution (PBS), and fixed with 4% paraformaldehyde at 4 °C for 24 h. Organ of Corti surface preparation was performed under a stereoscopic microscope . The cochlea was stained with phalloidin  and rinsed three times with PBS for 5 min each. Hair cells were observed by z-stacking with a confocal microscope . Three rows of outer hair cells (OHC) were counted within 200 μm per turn. Two separate locations per turn were used for measurements. If hair cells were empty or absent in photomicrographs, they were considered non-viable.On PID 49, the animals were anesthetized, and the middle ear was harvested, perfused with 4% paraformaldehyde in PBS, and fixed with 4% paraformaldehyde for 2 h at room temperature. After decalcification with 0.1 M ethylenediaminetetraacetic acid (pH 7.4) for 3 weeks, the bullae were embedded in paraffin wax, cut into 5-μm-thick sections, and stained with hematoxylin and eosin. The location of the TM was identified in a consistent manner by locating the malleus head and its fibrous connection to the TM. The mucosa at the base of the bulla (BB) was evaluated. Using a light microscope , the thicknesses of the TM and BB mucosa were measured using DP2-BSW software (Olympus).p-values < 0.05 were considered to denote statistical significance.Statistical analysis was performed using SPSS software . Continuous variables are expressed as means ± standard deviations. The Mann–Whitney U-test was used to compare outcomes. A non-parametric Mann–Whitney U test was used because the data were not normally distributed, and the sample size was smaller than 20 in each group.


South Africa has a high prevalence of co-existing tuberculosis and HIV. As ototoxicity linked to the treatments for these conditions occurs with concomitant exposure to other ear toxins such as hazardous noise exposure, it is important to investigate the combination impact of these toxins. Limited published evidence exists on the co-occurrence of these conditions within this population.The objective of this study was to compare the hearing function of gold miners with (treatment group) and without (non-treatment group) the history of tuberculosis treatment, in order to determine which group had increased risk of noise induced hearing loss. Furthermore, possible influence of age and HIV in these two groups was examined.A retrospective record review of 102 miners’ audiological records, divided into two groups, was conducted, with data analyzed both qualitatively and quantitatively.Findings suggest that gold miners with a history of tuberculosis treatment have worse hearing thresholds in the high frequencies when compared to those without this history; with evidence of a noise induced hearing loss notch at 6000 Hz in both groups. Pearson's correlations showed values between 0 and 0.3 (0 and −0.3) which are indicative of a weak positive (negative) correlation between HIV and hearing loss, as well as between hearing loss and age in this population.Current findings highlight the importance of strategic hearing conservation programs, including ototoxicity monitoring, and the possible use of oto-protective/chemo-protective agents in this population. Numerous health challenges plague the gold mining industry within South Africa, including occupational noise induced hearing loss (NIHL) as well as occupational lung diseases such as tuberculosis (TB).The mining industry represents 8% of South Africa's Gross Domestic Product (GDP), and is arguably one of the country's most important industries.8In 2012, AngloGold Ashanti reported that there were 446 new cases of TB amongst their employees.TB within the South African gold mining industry can be seen as a major health concern. This concern highlights the need to describe hearing functions of gold miners with and without the history of TB treatment, hence the current study.Specific objectives of this research were:(1)To describe and compare the hearing function of a group of gold miners with a history of TB treatment (treatment group) to that of a group without the history (non-treatment group) – both groups exposed to noise.(2)To establish a possible relationship that age and HIV might have on hearing function of these two groups of miners.A quantitative cross-sectional retrospective record review design was adopted, through data obtained from 102 (204 ears) participant medical files where hearing function of noise exposed miners with a history of being previously re-treated for TB was compared to those with no history of TB treatment, to establish who had increased risk for noise induced hearing loss. Purposive sampling was used to select the participant files who met the inclusion criteria for the study. However, because one of the aims of the study was to attempt to establish the possible impact that history of TB treatment may have on hearing function in miners, it was crucial to apply a set of participant inclusion criteria. Given the fact that a paucity of published research on this aspect of occupational noise induced hearing loss in South Africa exists, the researcher believed that it was crucial to have oversight over variables which could confound the results of the study . Hence the inclusion criteria listed below.Each group of gold miners comprised of 51 participant files (102 ears each), and participants were matched for age, gender, and years of exposure to occupational noise.The participant inclusion criteria were:Positive/negative history of past TB infection and past TB re-treatment for the treatment and non-treatment groups respectively. The treatment group only included participants’ audiograms post the re-treatment phase but matched with years of exposure and noise exposure in the non-treatment group.Age between 18 and 56 years.Available serial audiologic monitoring records.Participants with other possible causes of hearing loss such as middle ear pathology, history of meningitis, syphilis, use of chemotherapeutic agents, recreational noise exposure, etc., were excluded.Prior to commencement of the study, permission to conduct the research project was secured from the University's Medical Human Research Ethics Committee (Protocol n° M140331). Moreover, the study was performed in accordance with the ethical standards laid down in the 2012–2013 World Medical Association's Declaration of Helsinki. Permission to conduct the study at the mine was obtained from mine management and from the relevant heads of departments. Once permission was obtained and ethical clearance secured, the researcher conducted detailed record reviews at the research site.In order to ensure research reliability, controls were exercised pertaining to participant variables, parameters pertaining to the records reviewed and data analysis procedures employed. Over and above conducting record reviews, utilizing an independent rater during data analysis, a pilot study was also conducted to ensure reliability and validity of the data capturing tool.t-test was done in order to identify the difference in hearing status between the two groups. Next, the two-sample, one-tail t-test was performed to determine if HIV had an impact on hearing status. Lastly, Pearson's correlations were done in order to identify possible relationships between age and hearing loss, as well as between HIV and hearing loss. Correlation coefficients are expressed as values between +1 and −1; with +1 indicating a perfect positive correlation, while 0 indicates no correlation. Values between 0 and 0.3 (0 and −0.3) indicate a weak positive (negative) correlation; while values between 0.3 and 0.7 (−0.3 and −0.7) indicate a moderate positive (negative) correlation. Values between 0.7 and 1 (−0.7 and −1) indicate a strong positive (negative) correlation.To analyze data, the mean and standard deviation were calculated for each group. The mean scores were calculated for age, each PT (pure tone) frequency in each ear, as well as the PLH (percentage loss of hearing). Then, a two-sample, one-tail The sample  containeOn analysis of the mean hearing thresholds on audiograms , resultsThe thresholds in the low and mid frequency range  are better than 25 dB  in both groups.t-test used to determine if there was a difference in the hearing status between the two groups indicated that most of the cases rejected H0, implying that there was a statistically significant difference in hearing ability between both groups at all frequencies tested except at 250 Hz bilaterally. This finding was for all frequencies tested bilaterally (except 250 Hz). When both ears scores were combined, statistically significant differences were found at all frequencies, except at 250 Hz .Results from the two sample one-tailed t-test used to identify if HIV had an impact on hearing status in the current sample indicated that H0 was not rejected at any of the frequencies included, implying that HIV did not have an impact on the hearing status of gold miners in this population.Results from the two-sample one-tailed Pearson's correlations were done in order to identify possible relationship between age and hearing loss, as well as HIV and hearing loss in both groups. Values between 0 and 0.3 (0 and −0.3) were obtained; and these are indicative of a weak positive (negative) correlation  was seen in both groups with, however, additional high frequency hearing loss in the treatment group seems to confirm that exposure to TB treatment worsens hearing function in this noise-exposed population. This finding supports the documented evidence that TB treatments have a direct effect on an individual's hearing; and that concomitant exposure to ototoxic medications and noise imposes synergistic effects on the ear; thereby compounding the presenting hearing symptom.According to Valente et al.,th and 6th decade of life.The lack of correlation between age and hearing loss in both groups as a finding is not surprising. In the current study, the average age of gold miners without a history of TB treatment was 40.2 yrs and 45.4 yrs for those with a history of treatment. The natural deterioration of hearing with age becomes a factor during approximately the 5Current findings underscore the need for audiological involvement in the monitoring of hearing function in miners in this population. This need appears to be more pressing in miners with a history of TB treatment since these miners seem to present with worse hearing than those in the non-treatment group. Furthermore, the fact that HIV raises individual susceptibility to TB, as confirmed in the current study where 70% of participants with a history of TB treatment had HIV, means increased demand for audiology services in the South African noise exposed mining population who have TB and are HIV positive.Findings from this study are based on a relatively young age group of miners where presbycusis was not a confounding factor. The aging population of miners, in the presence of high prevalence of TB and HIV, raises more implications for audiological and otological clinical service delivery in this population. This study therefore, raises important implications about hearing conservation programs for miners with a history of TB in this context. Adherence to the use of personal hearing protection devices within a carefully run ototoxicity/noise monitoring programmer, as well as use of ototoxic-protective/chemo-protective agents in the miners who are on TB medication, are important considerations, particularly since use of personal hearing protection devices cannot be guaranteed.The author declares no conflicts of interest.


In vitro, we observed that transcription factor NF-κB, Th1/Th2 type cytokines  and the ability of catching H9N2 IAIV antigen were significantly up-regulated when H9N2 IAIV along with TLR ligands  and imiquimod, alone or combination) in duck MoDCs. Also, the best enhancement effects were showed in combination of pam2CSK4, poly (I:C) and imiquimod group, whereas IFN-α showed no significant enhancement in all experimental groups. In vivo, the results demonstrated that the percentages of CD4+/ CD8+ T lymphocytes, the levels of Th1/Th2 type cytokines and H9N2 HI titers were significant enhanced in combination of pam2CSK4, poly (I:C) and imiquimod group. However, pam2CSK4 alone or combining with imiquimod showed no enhancement or additive effects on Th1 cytokines (IFN-γ and IL-2), Th2 cytokines (IL-6) and HI titers in Muscovy duck, respectively. Taken together, our results concluded that not all TLR ligands showed enhancement of immune responses to H9N2 IAIV in duck. The combination of poly (I:C), imiquimod and pam2CSK4 that can be an effectively adjuvant candidate for H9N2 AIVs inactivated vaccine in duck, which provide novel insights in explore waterfowl vaccine.Current commercial H9 avian influenza viruses (AIVs) vaccines cannot provide satisfactory antibody titers and protective immunity against AIVs in duck. Toll like receptors (TLR) ligand as AIVs adjuvants can activate dendritic cells to improve immune responses in multiple animals, while the studies were absent in duck. Therefore, we investigated TLR ligands pam2CSK4, poly (I:C) and/or imiquimod enhance immune responses to inactivated H9N2 avian influenza antigen (H9N2 IAIV) in peripheral blood monocyte-derived dendritic cells (MoDCs) and duck.  Because of duck infected with H9N2 avian influenza viruses (AIVs) are generally overlooked due to the lack of clinical symptoms. Since 1994, H9N2 virus was first isolated in chicken in China, H9N2 subtype quickly spread to most areas of China and replaced H5N6 and H7N9 as the dominant AIVs subtype in duck in recent year, moreover, it is considered to be a potential candidate for a future pandemic . The strToll like receptors (TLR) play crucial roles in humoral and cellular adaptive immune responses –9. Afterin vitro and the use of TLR ligands combinations to enhance antibody responses in vivo for AIVs in duck.Previous studies have shown that some TLRs ligands, such as TLR2 ligand pam2CSK4, TLR3 ligand poly (I:C) and TLR7 ligand imiquimod have been explored as potential adjuvant for the influenza vaccine in chicken, mice and so on, respectively ,14–16. Ain vitro of dendritic cells and in vivo of Muscovy duck. Our experiments may provide novel insights to further explore in AIVs inactivated vaccine development in duck.Hence, in the present study, we aimed to evaluate the immunomodulatory effect and mechanism of TLR2 ligand pam2CSK4, TLR3 ligand poly (I:C) and TLR7 ligand imiquimod, alone or in combination, to inactivated H9N2 avian influenza antigen 50 at 108/0.1 mL) was propagated in 10-day-old embryonated SPF chicken eggs (Guangdong Enterprise Key Laboratory of Biotechnology R&D of Veterinary Biologics). Virus titers were measured following the method of Reed & Muench as described previously [H9N2 AIVs ) was obtained from the Guangdong Enterprise Key Laboratory of Biotechnology R&D of Veterinary Biologics. Briefly, the H9N2 vaccine candidate virus  was raised in an isolator to allow the animals to eat and drink normally. These ducks were raised for two weeks to adapt to their living environment. All duck serum samples were confirmed to be free of any AIV infection in the study. Adjuvant of TLR2 ligand pam2CSK4, TLR3 ligand poly (I:C) and TLR7 ligand imiquimod were purchased from InvivoGen .−ΔΔCt (where Ct is threshold cycle) method and shown as fold changes relative to the level for the control group. The amplification program was 95°C for 30 s, followed by 40 cycles with 5 s at 95°C for denaturation, 30 s at 55°C for annealing, and 30 s at 72°C for extension. After the amplification phase, melting curves were routinely performed to confirm the presence of a single PCR product.The total RNA of test samples were extracted and reversely transcribed to cDNA as previously study described . The rea7/mL in six-well plates at 37°C with 5% CO2 for 7 days to make cells differentiate into MoDCs. Half of the culture medium was removed with the replacement by equal volume of fresh medium every three days. 7 days later, most of cells showing a single or clustered non-adherent, irregular shape, veiled and/or dendrites  of healthy duck. In brief, the peripheral blood was collected from 2-week old healthy ducks (n = 3) by sterile vessels containing EDTA anticoagulant. PBMCs were isolated according to the manufacturer’s instructions of chicken peripheral blood lymphocyte isolation kit . Subsequently, PBMCs were cultured in RPMI-1640 medium containing 10% FBS, 100 IU/mL penicillin, 100 μg/mL streptomycin and stimulated with 20 ng/mL recombinant chicken IL-4 , 40 ng/mL recombinant chicken GM-CSF  at a density of 1× 10endrites . In addiendrites .7 EID50 H9N2 IAIV per well, respectively. For group g: MoDCs were treated with 1× 107 EID50 H9N2 IAIV alone per well. For group h: MoDCs were cultured with RPMI-1640 and used as mock control. Duck MoDCs were harvested at 6 and 12 h post-stimulation (n = 3/group/time point) to determine the relative mRNA expressions of TLR signaling pathway key node proteins , Th1-type cytokines (IL-2 and IFN-γ), Th2-type cytokines (IL-6), proinflammatory cytokines (IFN-α and TNF-α) and cell surface molecules (MHC-I and MHC-II). Quantitative real-time PCR primers used in this study were listed in Duck MoDCs were divided into eight groups: Group a: pam2CSK4-H9N2 IAIV group, Group b: poly(I:C)-H9N2 IAIV group, Group c: imiquimod-H9N2 IAIV group, Group d: pam2CSK4-imiquimod-H9N2 IAIV group, Group e: poly (I:C)-imiquimod-H9N2 IAIV group, Group f: pam2CSK4-poly (I:C)-imiquimod-H9N2 IAIV group, Group g: H9N2 IAIV group and Group h: RPMI-1640 group. For groups a–f: MoDCs were treated with pam2CSK4 (10 μg/mL), poly (I:C) (20 μg/mL) and/or imiquimod (5 μg/mL) along with 1× 10The MoDCs were harvested at 12 h post-stimulation (n = 3/group) to determine the function of MoDCs phagocytose H9N2 IAIV. In brief, MoDCs were fixed and permeability using Cytofix/Cytoperm according to the manufacturer’s instructions . MoDCs were then stained with Rabbit FITC-H9N2 HA protein monoclonal antibody  at 4°C for 1 h. Then washed twice with PBS, and re-suspended in 4% paraformaldehyde for flow cytometry . The data were analyzed using the Flowjo7.6 software.+ and CD8+ T lymphocytes, while the other part of spleen tissue was stored in liquid nitrogen for detecting cytokines mRNA expression.Two-week-old white Muscovy duck (n = 40) were allotted randomly to one of eight groups (n = 5/group) as showed in + T and CD8+ T cells in splenic lymphocytes of duck. The duck spleen lymphocyte was separated and then incubated with mouse anti-duck CD4 or CD8 antibody  1:100 at 4°C for 1 h. After washing twice with washing liquid, FITC-Sheep anti-mouse/rat IgG (H+L)  secondary antibody 1:1000 was added and incubated in darkness for 1 h. Then washed twice and re-suspended in 4% paraformaldehyde. The expressions of different cell surface markers were analyzed by flow cytometry.In order to evaluate the percentages of CD4TLR ligands induced cytokines mRNA expression in spleen tissue was evaluated at 21 dpv using qPCR to determine T cell function in the experimental ducks. In brief, isolation of spleen totals RNA and synthesis of cDNA according to the manufacturer’s instructions. Expression levels of mRNA of cytokines  and immune-associated genes  were analyzed by qPCR. Quantitative real-time PCR primers used were listed in P-values were less than 0.05.Statistical analysis was performed used using GraphPad Prism 6 software . Data are shown graphically as the geometric mean of the fold change plus the standard error of the mean (SEM). Differences in the expression level of immune-related genes in duck MoDCs and spleen tissues were analyzed using two-way ANOVA followed by Bonferroni’s test. Data from the HI levels and flow cytometry analysis were analyzed by one-way ANOVA with Tukey’s post-hoc test. The results were considered statistically significant when All animal experiments were carried out in strict accordance with the guidance of both of the Centers for Disease Control and Prevention’s Institutional Animal Care and Use Committee and the Association for Assessment and Accreditation of Laboratory Animal Care International. The protocol was approved by the Committee on the Ethics of Animal Experiments of Animal Biosafety Level 3 Committee of South China Agricultural University. Surgery and euthanasia was performed under anesthesia with sodium pentobarbital solution (100 mg/kg body weight) via intravenous route to minimize suffering.The mRNA levels of MyD88, TRIF and NF-κB were measured in duck MoDCs which stimulated with pam2CSK4, poly (I:C) and/or imiquimod along with H9N2 IAIV for 6 h and12 h by qPCR. In H9N2 IAIV, pam2CSK4-H9N2 IAIV and pam2CSK4-poly (I:C)-H9N2 IAIV groups, the mRNA levels of TRIF and MyD88 remained at the basal level .The mRNAIn vivo, the mRNA expressions of Th1-type cytokines (IL-2 and IFN-γ), Th2-type cytokines (IL-6), proinflammatory cytokines (IFN-α and TNF-α) and cell surface molecules (MHC-I and MHC-II) were up-regulated in almost all TLR ligand(s) -inactivated H9N2 AIV groups compare with inactivated H9N2 AIV group at 6 h post stimulation (P>0.05). As showed in P<0.05). As showed in P< 0.01).mulation . The higP<0.01).But, there was no statistical difference between combination of the pam2CSK4 and imiquimod to pam2CSK4 or imiquimod alone in the positive rates of MoDCs catching H9N2 IAIV antigen (P>0.05).Flow cytometry was conducted to evaluate the capability of MoDCs to phagocytose H9N2 AIV antigen at 12 h post-stimulation. Positive rates of MoDCs catching H9N2 IAIV antigen in RPMI-1640 mock control group, H9N2 IAIV group, pam2CSK4-H9N2 IAIV group, poly (I:C)-H9N2 IAIV group, imiquimod-H9N2 IAIV group, pam2CSK4-imiquimod-H9N2 IAIV group, poly (I:C)-imiquimod-H9N2 IAIV group and pam2CSK4-poly (I:C)-imiquimod-H9N2 IAIV group were 1.5%, 9.1%, 14.5%, 16.2%, 14.1%, 13.8%, 15.6% and 18.9%, respectively . Our resP< 0.05). The highest HI titer was seen in pam2CSK4-poly (I:C)-imiquimod-H9N2 IAIV group (average titer = 9) and which showed significant additive or synergistic effects on HI titers compared to the other group (P< 0.01). However, almost no significant difference was observed between the ducks that received imiquimod alone to that received combination of imiquimod and pam2CSK group (P>0.05).Humoral immune response was assessed by HI test on serum samples collected at 21dpv. The mean HI titers of RPMI-1640 mock control group, H9N2 IAIV group, pam2CSK4-H9N2 IAIV group, poly (I:C)-H9N2 IAIV group, imiquimod-H9N2 IAIV group, pam2CSK4-imiquimod-H9N2 IAIV group, poly (I:C)-imiquimod-H9N2 IAIV group and pam2CSK4-poly (I:C)-imiquimod-H9N2 IAIV group were 0.2 log2, 4.5 log2, 5.2 log2, 5.4 log2, 5.8 log2, 6 log2, 6.8 log2 and 9 log2, respectively . The HI P<0.05); ab means has no significant difference between adjacent a or b (P>0.05).Serum samples (5 ducks per group) were collected at 21-day post-vaccination. Antibody titers were determined using the HI assay with 4 HA units of the A/Chicken/Jiangsu/DHN12/2013 H9N2 AIV. The HI titer is expressed as the log2 form. Data were indicated as mean ±SEM. Different letters (a~c) mean significant difference (P< 0.05). However, the highest mRNA relative expression levels of IFN-α was showed in poly (I:C)-imiquimod-H9N2 IAIV group (P< 0.05).The relative expression level of MHC-II and CD4 mRNA in most TLR ligand(s)-H9N2 IAIV groups  was significantly up-regulated compare to H9N2 IAIV group -H9N2 IAIV group, imiquimod-H9N2 IAIV group and pam2CSK4-poly (I:C)-imiquimod-H9N2 IAIV group were analyzed at 21 dpv, the results were 1.8% and 2%, 11% and 13.1%,20% and 18.5%, 21.8% and 18%, 31% and 14%, 21.9% and 20.9%, respectively (+ and CD8+ T lymphocytes were in imiquimod-H9N2 IAIV group and pam2CSK4-poly (I:C)-imiquimod-H9N2 IAIV group, respectively. As the results shown in + T lymphocytes in imiquimod-H9N2 IAIV group, percentages of both CD4+ and CD8+ T lymphocytes in splenocytes of duck immunized with pam2CSK4-H9N2 IAIV, poly (I:C)-H9N2 IAIV, imiquimod-H9N2 IAIV and pam2CSK4-poly(I:C)-imiquimod-H9N2 IAIV was significantly increased when compared with H9N2 AIV antigen alone (P< 0.05).Given that the HI titers of pam2CSK4-imiquimod-H9N2 IAIV group and poly (I:C)-imiquimod -H9N2 IAIV group were lower than pam2CSK4-poly (I:C)-imiquimod-H9N2 IAIV group in duck. The percentages of CD4ectively . The higH9N2 AIVs continue to circulate in duck and became the dominant subtype AIVs after 2014, partly reasons of the current inactivated vaccines exhibited lower level of influenza antibody and poor cell mediated immune response in duck when compared to chicken ,5. AlthoTLR3 signals through the adaptor protein TRIF while TLR2/7 signals through the adaptor protein MyD88, but share some common regulators such as NF-κB ,11. The in vitro was shown to increase production of IFN-γ [Cytokines play an important role in the body’s immune response. Poly (I:C) and imiquimod have been demonstrated to induce up-regulation of IFN-α in both birds spleen cells and pig MoDCs ,15,19. Hof IFN-γ .However, the synergistic effect was not seen in all combination groups. For example, the mRNA levels of cytokines in duck MoDCs of pam2CSK4-imiquimod-H9N2 IAIV group showed no synergistic enhancement effect compares to imiquimod-H9N2 IAIV group. It is could be that TLR ligands combinations with similar signaling pathways may be have a certain thresholds ,27–29. IIn vivo experiment, the highest levels of IFN-γ, IL-6 and HI titers were showed in pam2CSK4-poly (I:C)-imiquimod-H9N2 IAIV group (P< 0.05). It seems that combination of pam2CSK4, poly (I:C) and imiquimod with H9N2 IAIV could induce a mixed Th1-/Th2-like response and given complementary effect on serum titers in duck [+ and CD8+ T lymphocytes of duck splenocytes were higher in pam2CSK4-poly(I:C)-imiquimod-H9N2 IAIV group. Highly expression and production of these proteins in the spleen are crucial for the development of immune responses [ in duck –32. Alsoesponses ,33–35. Tesponses ,36.Significantly, HI titers in pam2CSK4-imiquimod-H9N2 IAIV group showed no difference to imiquimod-H9N2 IAIV group, but lower than poly (I:C)-imiquimod- H9N2 IAIV group, indicating that combination of poly (I:C) and imiquimod could produce stronger immune response than combination of pam2CSK4 and imiquimod in duck. Our data showed that both TLR ligands together did not always give any complementary effect on serum titers in all animals. Similarly, vaccination experiments in chickens using an inactivated H9N2 AIV vaccine have shown that combining TLR5 and TLR21 ligands as adjuvants does not lead to synergistic or additive increases in antibody-mediated immune responses compared with administering either ligand alone .in vitro experiments, which need further study. It is noteworthy that vaccine administered by intraperitoneal was used in this study, whereas intramuscularly or subcutaneously was used in previously studies. However, previous studies have shown that intraperitoneal imiquimod can augment and accelerate the immune response, and improved vaccine efficacy for H7N9 AIVs in a mouse model [Previous works have shown that pam2CSK4 and poly (I:C) could effective enhancing systemic antibody mediated immune responses when administered intramuscularly in chicken and duck when compared with vaccines containing only inactivated influenza virus, respectively ,16,37. Ise model . We specIn summary, H9N2 IAIV along with pam2CSK4, poly (I: C) and imiquimod alone or in combination can significantly enhance cell-mediated immune responses and ability of presenting inactivated H9N2 antigen in duck MoDCs, and importantly the interferon effect induced maybe caused by IFN-γ, not IFN-α. Moreover, we have demonstrated that combination of pam3CSK4, poly (I:C) and imiquimod is an effective adjuvant system capable of boosting antibody responses and inducing T cell activation when co-administered with H9N2 IAIV intraperitoneal Muscovy duck. It is also shown that combining pam2CSK4 with imiquimod compare to imiquimod alone did not have significant additive and synergistic effects on antibody responses of H9N2 IAIV in Muscovy duck. Nevertheless, future experiments should examine the immunity protective efficacy to H9N2 AIVs with different combinations of TLR ligands vaccine by different vaccination strategies in duck to further verify the conclusions of this paper.S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.