Need for recovery is a predictor of work stress and health problems, but its underlying factors are not yet well understood. We aimed to identify hearing-related, work-related, and personal factors influencing need for recovery in hearing-impaired employees.N = 294) that were referred to the Amsterdam University Medical Center between 2004 and 2019. Routinely obtained healthcare data were used, including a survey and hearing assessments. A directed acyclic graph was constructed, revealing the hypothesized structure of factors influencing need for recovery as well as the minimal set of factors needed for multiple regression analysis.We retrospectively identified hearing-impaired employees , self-perceived listening effort , personal adaptations scale score , and having a moderate/poor general health condition . Although degree of hearing loss was associated with self-perceived listening effort, the direct association between degree of hearing loss and need for recovery was not significant.Four variables were included in the regression analysis. In total, 46.1% of the variance in need for recovery was explained by the factors feeling that something should change at work (The results suggest that the way employees perceive their hearing loss and how they cope with it directly influence need for recovery, rather than their measured degree of hearing loss. Additionally, general health condition was found to be an independent factor for need for recovery. The results should be confirmed by future, longitudinal research. Hearing loss is a common occupational malady . Factors potentially influencing NFR were derived from hearing assessments performed at the hospital and a hearing survey that was completed at home. All variables were derived from patient files.We retrospectively identified patients referred to Amsterdam UMC’s ENT-Audiology department (location AMC) by their occupational physician. All patients were thus referred from occupational healthcare. Eligible patients visited the hospital between 2004 and 2019, were aged between 18 and 67, underwent pure tone and speech audiometry, and completed the hearing survey prior to their hospital visit. Patients were included regardless of the cause of their hearing loss. For patients with multiple referrals, the data were included belonging to the first referral with a completed questionnaire and hearing evaluation. To prevent bias, patients were excluded if they were referred to the hospital for a fitness for job assessment by their employer. The reason is that hearing loss complaints might be experienced or reported differently if continuation of the job depends on it. All patients received a letter with information about the study. Because of the retrospective study design, an opt-out procedure was performed.n = 283) were not eligible for the study, as were patients older than 67 (n = 2). Further, patients were excluded that declined to participate (n = 6) or had an incomplete patient file, specifically missing tone audiometry (n = 4), missing survey (n = 20), or incomplete survey (n = 37).A total of 646 patients were identified of being referred to the ENT-Audiology department by their occupational physician Fig. . PatientThe data were collected retrospectively by review of patient files. Outcomes of the hearing survey and the hearing assessments were entered into Castor, an electronic database  were performed in a sound-isolated booth using calibrated clinical audiometers (AC40 and Decos audioNigma) and TDH 39 headphones. According to the hospital protocol, pure tone thresholds for air and bone conduction were reported in decibel (dB) hearing level (HL) at frequencies 250 Hz, 500 Hz, 1000 Hz, 2000 Hz, 4000 Hz, and 8000 Hz, with adequate masking (if necessary). For a healthy, normally hearing individual, hearing thresholds up to 25 dB HL are regarded as normal (Martin and Champlin Speech reception in continuous noise was assessed in a free field setting (S0N0) using everyday Dutch sentences developed by Plomp and Mimpen  or the sPrior to the hospital visit, patients completed an extensive hearing survey to investigate personal and environmental factors that potentially influence NFR. This survey consisted of three questionnaires and additional questions.Three scale scores were derived from the QEEW, a generic questionnaire on psychosocial workload and work stress , we have calculated a sum score of these six items, further considered as self-perceived listening effort. This score can range between 0 and 18.We did not only investigate the occurrence of the six hearing-related job activities, but also the effort they take. Since these six additional questions on the effort of hearing have good internal consistency (Other questions derived from the Amsterdam Checklist for Hearing and Work (ACHW) include the number of working hours a week  and fulfilling managerial tasks (dichotomous). All patients were asked whether they fulfilled managerial tasks, because managerial activities can be embedded in many professions and require specific skills that might appeal to hearing acuity, such as organizational and social skills Whitley .To investigate the coping strategies, the CPHI was used. The CPHI aims to distinguish between adequate and inadequate coping behavior  was manually performed with all variables directly related to NFR. As a result of the strategy used to select factors for the analysis, the model was unadjusted for other factors. We checked for interaction effects with the use of hearing aids or not with all variables in the analysis, because the relationship between objective and subjective factors might be different for employees that wear hearing aids. If an interaction term was not found to be significant (p > 0.05), it was removed. Data organization and statistical analysis were performed using the Statistical Package for Social Sciences (SPSS) version 25.0 (Armonk New York USA). The critical value of significance was 0.05 for all statistical analyses.Linear regression with a forward stepwise selection method , were included the study , administrative job (19.4%), doctor/nurse (10.2%), and managerial jobs (9.2%). Many patients with and without managerial jobs reported to fulfil managerial tasks (88.4%). The mean number of working hours per week was 33.6 (SD 8.7). The mean score for NFR was 54.94 (SD 34.12). In 55.8% of the participants, the NFR score was above 54, indicating an increased risk at occupational and health problems , self-perceived listening effort (r = 0.527), personal adaptations (r = − 0.456), and general health condition (r = 0.453). Two of these variables did also directly influence the secondary outcome measure self-perceived listening effort, respectively, feeling that something should change at work (r = 0.390), and personal adaptations (r = − 0.442). Self-perceived listening effort was also directly influenced by BHI (r = 0.318) and auditory work demands (r = 0.413). The hearing assessment outcomes did not significantly correlate with NFR, including BHI , maximum discrimination score , and SNR . All correlations between the hypothesized factors and the primary and secondary outcome are shown in Table The presence of tinnitus, age, and the educational level were not directly or indirectly associated with NFR and self-perceived listening effort. Consequently, these factors were not included in the directed acyclic graph. Figure  r = 0.45. Two of p < 0.001) of the NFR. Feeling that something should change at work, self-perceived listening effort, the personal adaptations scale score, and having a moderate/poor general health condition were significantly related to higher NFR. In the secondary regression analysis (Table p < 0.01) of the self-perceived listening effort. Feeling that something should change at work, BHI, auditory work demands, and the personal adaptations scale score were significantly related to self-perceived listening effort. In both analyses, there were no significant interaction effects.The results of the primary regression analysis Table  indicateis Table , the fouThe aim of this study was to identify factors influencing NFR and listening effort in hearing-impaired employees. Four factors were shown to directly influence NFR and four factors were shown to directly influence self-perceived listening effort.r = 0.527) in the correlation analysis. In contrast, no significant associations were observed between the hearing test outcomes and NFR, including BHI, maximum discrimination score, and the critical SNR. While tinnitus has earlier been shown to be associated with NFR , the personal adaptation score was directly related to self-perceived listening effort and NFR, but the communication strategy score was not. Other studies report an association between communication strategies used and NFR (de Vries et al. In line with de Vries et al.  and MachIn addition to explore the influence of coping behavior, several other questions were included to assess the influence of personal factors. We observed that the factor feeling that something should change at work was moderately associated with NFR, as well as with self-perceived listening effort. The question “Do you feel something should change in your work situation” may grasp a feeling of frustration at the workplace, that was earlier associated with NFR in seafarers (Bridger et al. In line with the previous studies (Gommans et al. Other work characteristics did neither influence NFR nor self-perceived listening effort. First, we expected a positive association between the number of working hours and NFR (Jansen et al. We have derived work-related factors from the QEEW and ACHW, because these questionnaires are routinely performed in the ENT-Audiology clinic. Therefore, we may have missed other work-related factors that influence NFR in hearing-impaired employees, such as job control, job demand, and social support. The included scale score of collegial support does not reflect all aspects of the construct social support, since this construct also refers to helpful social interactions from supervisors (Nachtegaal et al. Some study limitations should be noted. First, the retrospective character of the study implicates a risk for measurement bias. For example, the hearing tests were performed by multiple clinicians following clinical protocols, rather than a research protocol, which may have caused differences in measurement settings. Despite this limitation, the four identified factors accounted for 46.1% of the variance in NFR and 43.1% of self-perceived listening effort. Second, the cross-sectional design is a limitation of this study, since it does not allow drawing conclusions about causality. Constructing a directed acyclic graph allowed for visualization of the relationship of a broad spectrum of factors influencing NFR. Since the evidence on factors influencing NFR in hearing-impaired employees was limited, this explorative method is considered to be appropriate. A prospective study is needed to verify and validate the findings of this study. To gain further understanding in the difficulties of hearing-impaired employees and the efficacy of intervention strategies that aim to reduce these difficulties, future clinical trials are recommended to assess the efficacy of audiological, speech therapeutic, and social interventions on both self-perceived listening effort and NFR.This study provides a framework of factors associated with NFR in hearing-impaired employees, contributing to the understanding of occupational problems in this population. The results suggest that the way employees perceive their hearing loss and how they cope with it directly influence NFR, rather than their measured degree of hearing loss. Further, when assessing or evaluating NFR, an employees’ general health condition should be considered. These findings are relevant for clinicians and occupational physicians that perform diagnostics or intervention strategies for hearing-impaired employees. Also, the results may contribute to gain understanding in the working mechanisms of interventions that aim to prevent or cure occupational diseases in employees with hearing loss.


Triclosan (TCS) is one of the most common endocrine-disrupting chemicals (EDCs) present in household and personal wash products. Recently, concerns have been raised about the association between abnormal behavior in children and exposure to EDC during gestation. We hypothesized that exposure to TCS during gestation could affect brain development. Cortical neurons of mice were exposed in vitro to TCS. In addition, we examined in vivo whether maternal TCS administration can affect neurobehavioral development in the offspring generation. We determined that TCS can impair dendrite and axon growth by reducing average length and numbers of axons and dendrites. Additionally, TCS inhibited the proliferation of and promoted apoptosis in neuronal progenitor cells. Detailed behavioral analyses showed impaired acquisition of spatial learning and reference memory in offspring derived from dams exposed to TCS. The TCS-treated groups also showed cognition dysfunction and impairments in sociability and social novelty preference. Furthermore, TCS-treated groups exhibited increased anxiety-like behavior, but there was no significant change in depression-like behaviors. In addition, TCS-treated groups exhibited deficits in nesting behavior. Taken together, our results indicate that perinatal exposure to TCS induces neurodevelopment disorder, resulting in abnormal social behaviors, cognitive impairment, and deficits in spatial learning and memory in offspring. Triclosan (TCS) is widely used in consumer products as an antibacterial agent and is found in toothpaste, mouthwash, soap, deodorants, textiles, toys, and medical devices . TCS is Notably, adversity during early-life brain development is associated with an increased risk for the development of behavioral and psychiatric disorders, including depression, anxiety, and schizophrenia . In addi−8 M), TCS showed no effect on the number of primary dendrites compared to that in vehicle group  (A previous study suggested that exposure to TCS delays the development of motor neurons . As TCS le group B. The nule group C. Howeve 0.0001) D.−6 M), the length of primary dendrites in the TCS 10−6 M group was significantly decreased compared to that in the vehicle group    B. The nu 0.0300) C. In add−8 M group were no significant differences compared to those in the vehicle group (−6 M), the length of axons in the TCS-treated group was significantly decreased compared to that in the vehicle group   C.−6 M group compared to that in the vehicle group    F. Howeve 0.4174) G. Compar+ /DAPI cell was quantified. The percentage of BrdU+ cells in both TCS 10−8 M and TCS 10−6 M groups were lower than in the vehicle group   H,I. More M group H,I.+/DAPI cell was quantified. As expected, the percentages of caspase-3+ cells were markedly higher in the TCS 10−6 M group compared to the vehicle group   J,K. Addie groups K. In add+, Ki67+ and p-Histone-H3+ cells in the DG were determined. The number of BrdU+ cells was slightly decreased in the E18.5 TCS-treated groups compared to that of the vehicle group   (p-Histone-H3) at the DG were lower in the TCS-treated groups than in the vehicle group   area of TCS-treated and vehicle embryos by injecting pregnant females with BrdU at E18.5 and harvesting embryos 2 h later A. The nu 0.0001) A,B. In a 0.0120) A,C. More 0.0001) A,D. Therg groups A,D. +Ki67+/BrdU+) . However, TCS 10 mg/kg group showed no significant difference in the proportion of cell cycle re-entry compared to the TCS 100 mg/kg group  E,F      . Moreove 0.0008) . There w 0.0008) . Moreove0.0463)  . Additio 0.0075) . These r2,9 = 10.84, p = 0.0040)   . Additio 0.0106) . In addiTg), thyroperoxidase (Tpo), sodium-iodide symporter (Nis), monocarboxylate transporter 8 (Mct8) and thyrotropin receptor (Tshr) in the thyroid gland at PND 119 (Trhra), thyrotropin releasing hormone (Trh) in brain and thyrotropin releasing hormone receptor (Trhr) in pituitary gland (Dio2) in brain was markedly higher in TCS-treated groups compared to vehicle group (Mct8 and organic anion transporter family member 1C1 (Oatp1c1) were lower in brain of TCS-treated groups compared to vehicle group  groups  groups   . We first performed the Morris water maze test to assess the spatial learning and memory impairment of members of the TCS-treated groups. Compared to the vehicle group, both TCS-treated groups exhibited increased time to find the hidden platform during the acquisition phase  A. On tes) groups C. In add) groups D. Visual) groups B. But, t 0.0321) E. Additi 0.0259) F.We also assessed recognition memory by using a novel-object recognition test. The mice were explored to two identical objects (similar shape and color), and after 6 h, one of the objects was replaced by a new novel object (different shape and color from that of the old object) G.p = 0.0021) (p = 0.0545) and TCS 100 mg/kg; Familiar = 49.39 ± 1.82, TCS 100 mg/kg; Novel = 50.61 ± 1.82, )  H. In con0.6144)) H. FurtheNext, we used the three-chamber social test to assess social interaction and discrimination of social novelty for each mouse. In addition, analysis of the preference index for social behaviors allows the direct comparison of the social behaviors of the groups. In the sociability test, the mice explored the three-chamber apparatus with the presence of an unfamiliar mouse in one of the side-chambers A. p < 0.0001); TCS 10 mg/kg; Empty = 186.19 ± 11.16, Stranger I = 237.18 ± 16.23, ; TCS 100 mg/kg; Empty = 151.51 ± 7.40, Stranger I = 251.19 ± 13.90, )  (p = 0.0092)) (p = 0.5805); TCS 100 mg/kg; Stranger I = 183.92 ± 6.94, Stranger II = 212.21 ± 13.12, )  (Both the vehicle and TCS-treated groups spent significantly more time in the chamber containing the unfamiliar mouse (S1) than in the empty chamber (E) ) B. Analys 0.0001) C. In the0.0092)) A,D. Unli0.0549)) D. Compar 0.0015) E. Intere 0.0015) E. These 2,43 = 3.962, p = 0.0245)  and anxiety-like behavior (reflected by the time spent in the center zone). The TCS 100 mg/kg group spent significantly less time in the open field center zone compared to the vehicle group (F 0.0245) B. 2,43 = 13.75, p < 0.0001) . In addition, the TCS-treated groups showed no difference in velocity compared to vehicle group   C. Moreov 0.0001) C. Howeve 0.0001) C. There  0.0001) D between 0.4637) E. There  0.4637) D,E. Take2,34 = 0.4342, p = 0.5535)   F. There  0.5535) F. The fo 0.0522) G. These 2,33 = 9.665, p = 0.0005)  . These rIn recent years, there have been increasing concerns about the effect of EDCs on human health. Of special concern is the placental transfer of EDCs from mother to fetus; thus, fetuses and children are particularly vulnerable to the effects of EDCs ,24. UnsuTCS has been identified in the milk and plasma of nursing mothers in Sweden and Australia ,28. TCS In addition, as an EDC, TCS has endocrine-disrupting properties and may affect the regulation of estrogen, testosterone, and thyroid hormone levels in animal experiments. Some studies have shown that exposure to TCS reduces the level of thyroid hormone in rats , while oBoth human and mouse brain development are remarkably complex processes and begin during early gestation and continue into the postnatal period. Thus, in this study, TCS was introduced on E9.5 in mice to investigate its effects on brain development. At high dose, TCS showed a decrease in birth weight and the numbers of offspring mice compared to vehicle group. This suggests that TCS can have adverse effects on fetal development ,11. The Pax8/Nkx2-1 associated with the reduction of their transcriptional activity  which involved in the thyroid hormone synthesis and secretion in adult offspring mice. Importantly, the increased expression of Trh and Trhr indicated the reduced action of thyroid hormone in the hypothalamus-pituitary axis. In fact, Trh and Trhr mRNA are known to be down-regulated by T3 [Nis dysfunction impaired to the thyroid hormonogenesis in human and caused congenital goitrous hypothyroidism [Mct8 and Oatp1c1. Recently, Mct8 and Oatp1c1 double knockout mice showed hypothyroidism. Additionally, expression of Dio2, that is known to be regulated by T3 and T4 serum levels, was markedly increased in the brain of Mct8/Oapt1c1 double knock-out mice [Pax8/Nkx2.1 expression associated with their reduced transcriptional activity might contribute to the decreased expression of genes/proteins involved in the TH synthesis and secretion  [In an early study, thyroid hormone was shown to promote Bcl-2 expression and prevent apoptosis of early differentiating cerebellar granule neurons . Thyroided by T3 . Moreoveyroidism . In addiyroidism . In braiout mice . Furthernd Mct8) . Our datnd Mct8) . Moreovend Mct8) .To address the functional relevance of the morphological and biochemical changes in the TCS-treated groups, we examined cognitive, social, and emotional behaviors. In previous studies, TCS has been reported to impair hippocampal synaptic plasticity  and fetaIn conclusion, we have observed that exposure to TCS during the perinatal period disrupted mouse brain development by affecting neurogenesis and neurite growth. Consequently, the offspring mice from the TCS-treated dams showed impairments to memory, social behavior, and anxiety behavior. However, as an EDC, TCS may affect thyroid activities ,10. Idenn = 5 mouse/group, each mouse was maintained in each cage) and they chronically received different TCS levels by subcutaneous injection. Mice in the three groups were given 0.2 mL of corn oil with TCS concentrations of 0, 10 and 100 mg/kg every day from E9.5 to postnatal day (PND) 28.5. In mice, the dose of 10 mg/kg TCS exposure caused an equivalent urinary TCS level as those in the high-TCS abortion patients [Specific pathogen-free adults C57BL/6J male and female mice  were obtained from Samtaco . The mice were housed in polycarbonate cages under a controlled environment conditions as in previous work . After tpatients . In orde5 neuronal cells were plated in poly-D-lysine coated 24-well plates and cultured in Neurobasal medium/DMEM (1:1) with B27 supplement, penicillin, streptomycin and glutamine  at 37 °C in a humidified incubator with 5% CO2/95% air. The day of plating was considered day in vitro 0 (DIV 0). In recent study, the concentrations lie within the 0.4–64 nmol TCS/mg tissue protein levels may result from human dermal exposure to TCS-containing products. In addition, 0.8–2.8 nmol TCS/mg protein doses within cells/tissues induce adverse effects [−8 M. On DIV 1, cells were treated with vehicle controls  and TCS at a low concentration 10−8 M or 10−6 M. On DIV 4, neurons were harvested for quantification of axon and dendrite morphology. For proliferative experiments, neurons were incubated with 5-bromo-3-deoxyuridine  after 12 h treated with TCS. Then, neurons were harvest after 2–3h BrdU-treatment.Primary cortical neuron culture was performed as described previously . The pri effects . In mice effects . Thus, wAxons were identified with the axonal marker Tau1 and dendrites were identified with the dendritic marker MAP2. For axons: the longest axon was recognized as the primary axon; the remainder was considered axonal branches. The secondary axons grow branching point in the primary axon. The length of primary axon was determined starting from the base of an axon (the site attached to the soma) to the tip. For dendrites: the primary dendrites are the processes directly emerging from the soma. The secondary dendrites grow branching point in the primary dendrite. The total dendrite length, including primary dendrites and all dendritic branches. Measurements of axon and dendrite length were carried out using the ImageJ software .Neuronal cells were fixed in 4% formaldehyde (PFA) and then permeabilized with phosphate buffered saline (PBS) containing Triton X-100 (Sigma-Aldrich) (0.01% for cell). Then, neuron cells were blocked in PBS ++  + 0.25% Triton X-100) for 1h, followed by incubation in primary antibodies  at 4 °C overnight. For secondary staining, cells were incubated for 1h in secondary antibody solution  that contain 100 ng/mL 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich). Then cells were mounted in Flouro-Gel .BrdU staining was performed as described previously . NeuronaTotal RNA was extracted from whole brain, thyroid and pituitary gland of each mice by using Trizol reagents  according to the manufacturer’s protocols. cDNA synthesis was performed as previously described . QuantitBrain total protein content was extracted using Pro-prep solution  according to the manufacturer’s protocol. 100 g of protein was resolved by using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membrane  as previously described . Then, tAt 6 and 10 week-age, offspring mice were randomly selected to perform behavioral tests as described . On testThe open filed test performed in a large acrylic cube measuring by 50 cm tall, 60 cm wide with a white bottom. Briefly, mice were individually placed near the wall-side and allowed to freely move for 5 min to measure locomotor activity. The movement of mice was recorded and analyzed with EthoVision XT14 software . Time spent in the center zone (15 × 15 cm imaginary square), velocity and distance travelled was evaluated.The Morris water maze test was conducted in a circular pool , filled with water (25 ± 1 °C) and the water was made opaque by adding skim milk. The tank was divided into four equally sections . A circular platform  made of plexiglass was placed in the middle of a target quadrant (III) (12 cm from the pool’s edge and 1 cm below the surface of the water) with visual cues on the pool walls as spatial references. Mice received a two-phase training protocol for nine days: cue training (four days) and followed by spatial training (five days); four trials were performed per mouse per day and the escapes latency (time to find the hidden platform) in each trial was recorded. For each trial, the subject mice were gently placed into the water facing the wall from one of three quadrants , varied by day of testing. Then, mice were given 60 s to find platform and a trial completed when the mice had found the platform. If the mouse failed to find the platform within 60 s period, it was gently guided onto the platform by the experimenter and allowed to rest on it for 30 s. The mean of escape latencies (second) for 4 trials is represented for the learning result for each mouse. On day 10 (probe test day), the platform was removed from the pool, and the mice were left to search for the platform for 60 s. Videos were recorded and analyzed using EthoVision XT14, the time spent in the target platform and the number of crossing the platform were measured to indicate the memory results.The first, the subject mice were placed into the open field arena in the presence of two identical objects  and freely explored for 10 min. After 6 h, one of the objects was replaced by a novel object (different shape and color compared to old object) and the subject mice were placed inside the field to explore for another 10 min. The duration of time mice spent to interact with the novel and old objects (sniffing or exploring at distance within 2 cm of the object) were recorded. Videos were analyzed using EthoVision XT14.Each mouse was suspended on the edge of a shelf, 50 cm above the surface of a table. The subject mice were allowed to move for 6 min, and behavior was recorded by a camera. Videos were analyzed using EthoVision XT14. The duration of immobility was recorded in the last 5 min.Each mouse was gently placed into a glass cylinder  filled with water (25 ± 2 °C) to a depth of 12 cm. All mice were forced to swim for 5 min and the duration of immobility was recorded. Videos were analyzed using EthoVision XT14.The three-chambered social test was performed as follows . The thrThe nest-building behavior of the mice was assayed by assessing the nest quality after 12 h exposure to a nestlet of tissue cotton . Each mouse was placed in its one clean cage with a nestlet at 19:00. Nest-building ability was assessed according to a 5-point rating scale from 1 to 5 as follows . All micp values for each comparison are described in the results section. Each experiment in this study was performed blind and randomized. Animals were assigned randomly to the various experimental groups, and data were collected and processed randomly. The allocation, treatment, and handling of animals were the same across study groups. The number of mice and statistical detail for all behavioral assays are described in All statistical analyses were performed by applying two-way ANOVA, unpaired Student’s t tests for two population comparisons or one-way ANOVA (Bonferroni’s multiple comparison test). Data were randomly collected and analyzed using GraphPad Prism software . The results are presented as means ± SEM and the


We report a large family with four successive generations, presenting with a complex phenotype of severe congenital neutropenia (SCN), partially penetrant monocytosis, and hearing loss of varying severity.We performed whole exome sequencing to identify the causative variants. Sanger sequencing was used to perform segregation analyses on remaining family members.We identified and classified a pathogenic GFI1 variant and a likely pathogenic variant in MYO6 which together explain the complex phenotypes seen in this family.We present a case illustrating the benefits of a broad screening approach that allows identification of oligogenic determinants of complex human phenotypes which may have been missed if the screening was limited to a targeted gene panel with the assumption of a syndromic disorder. This is important for correct genetic diagnosis of families and disentangling the range and severity of phenotypes associated with high impact variants. Severe congenital neutropenia (SCN) was first described by Kostmann in 1956 in 14 individuals from 9 consanguineous families . It may ELANE, the gene encoding neutrophil elastase, are the most common cause of AD SCN. Growth factor independent 1 transcriptional repressor (GFI1) germline variants have been reported in four patients with neutropenia; inheritance patterns were consistent with an AD mode of inheritance .Fig.FigGATAGFI1 variants have not previously been associated with hearing loss, we explored additional genetic causes for the hearing loss phenotype observed in this family. Over 100 genes have been reported to underlie hereditary hearing loss. Analysis of the data for variants in genes associated with non-syndromic hearing loss  . Patients with SCN reportedly have a 21% cumulative incidence of myeloid malignancy after 10 years with the risk of leukemia being higher in patients requiring high doses of G-CSF, and is associated with acquired mutations in CSF3R and RUNX1 [SCN can be a pre-leukemic syndrome with evolution to leukemia recognized in patients with nd RUNX1 .GFI1 and ELANE [GFI1 variant carriers (aged 20–75 years) and 2 likely carriers (over 71 years) without progression to myeloid malignancy, suggesting that the GFI1 variant alone does not confer a high risk of leukemia development.To date, there have been no reports of MDS or AML in individuals with GFI1 variants alone  [MYO6) have been identified in AD and recessive hearing loss [Variants in myosin genes are known to be involved in several types of syndromic and non-syndromic hearing loss. Variants in myosin VI (ing loss , 13. Theing loss . The myoing loss . The I11ing loss . MiyagawGFI1 has also been reported to be essential for inner ear hair cell differentiation [MYO6, but carries the GFI1 variant, is an interesting case in this respect. Another curious observation is that family members with severe hearing impairment carry both variants while those with milder hearing problems only carry either of the variants. Though the MYO6 variant is most likely responsible for hearing loss in the family, it remains to be established if the GFI1 variant contributes to the phenotype.Intriguingly, ntiation , 16. HenGFI1 causing SCN, and MYO6 leading to hearing loss. Broader screening may be warranted in cases with complex disease presentations as polygenic inheritance could be missed if testing is restricted to specific gene panels. Although progression to leukemia has not been described to date in the limited number of individuals carrying germline pathogenic GFI1 variants, it remains to be established whether molecular monitoring for acquired variants should be considered as part of a risk management scheme.We have presented a family with a complex phenotype of SCN and hearing loss that can be attributed to AD variants in two genes, Additional file 1: Table S1. Neutrophil and monocyte counts of individuals where data was available. Table S2. Genes known to be mutated in congenital neutropenia. Table S3. Genes known to be mutated in non-syndromic hearing loss. Table S4. Computational predictions, variant annotation and classification.Additional file 2: Figure S1. Sequence conservation of mutated residues in GFI1 and MYO, across species.


People with hearing loss, particularly those who lose their hearing in adulthood, are at an increased risk of social isolation, mental health difficulties, unemployment, loss of independence, risk of accidents, and impaired quality of life. In the United Kingdom, a single third-sector organization provides hearing dogs, a specific type of assistance dog trained to provide sound support to people with hearing loss. These dogs may also deliver numerous psychosocial benefits to recipients. This has not previously been fully investigated.The study aims to evaluate the impact of a hearing dog partnership on the lives of individuals with severe or profound hearing loss.A 2-arm, randomized controlled trial will be conducted within the United Kingdom with 162 hearing dog applicants, aged 18 years and older. Participants will be randomized 1:1 using a matched-pairs design to receive a hearing dog sooner than usual (intervention arm: arm B) or to receive a hearing dog within the usual timeframe (comparator arm: arm A). In the effectiveness analysis, the primary outcome is a comparison of mental well-being 6 months after participants in arm B have received a hearing dog (arm A have not yet received a hearing dog), measured using the Short Warwick Edinburgh Mental Well-Being Scale. Secondary outcome measures include the Patient Health Questionnaire-9, Generalized Anxiety Disorder-7, and Work and Social Adjustments Scale. An economic evaluation will assess the cost-effectiveness, including health-related quality-adjusted life years using the EuroQol 5 Dimensions and social care–related quality-adjusted life years. Participants will be followed up for up to 2 years. A nested qualitative study will investigate the impacts of having a hearing dog and how these impacts occur.The study is funded by the National Institute for Health Research’s School for Social Care Research. Recruitment commenced in March 2017 and is now complete. A total of 165 participants were randomized. Data collection will continue until January 2020. Results will be published in peer-reviewed journals and at conferences. A summary of the findings will be made available to participants. Ethical approval was received from the University of York’s Department of Social Policy and Social Work Research Ethics Committee (reference SPSW/S/17/1).The findings from this study will provide, for the first time, strong and reliable evidence on the impact of having a hearing dog on people’s lives in terms of their quality of life, well-being, and mental health.International Standard Randomised Controlled Trial Number Registry ISRCTN36452009; http://www.isrctn.com/ISRCTN36452009DERR1-10.2196/15452 Around 5% of the world’s population currently experience disabling hearing loss, and this is estimated to nearly double by 2050 . In the In the United Kingdom, just one organization, Hearing Dogs for Deaf People  trains aprofile of their lifestyle and hearing dog–relevant needs. This profile is then released to the hearing dog training staff who liaise with the client advisor to identify matches between an applicant and the pool of hearing dogs coming toward the end of their training (aged approximately 18 months old). It can take between 1 and 3 years for a hearing dog to be provided to an applicant depending on the complexity of their needs. During the period of introducing the hearing dog to the recipient and the dog settling in their home, a series of strategies ensure the hearing dog attaches exclusively to the recipient. Apart from when the recipient is exercising the dog, the hearing dog wears a distinctive hearing dog jacket when taken outside the home. Hearing Dogs for Deaf People refers to the arrangement of a hearing dog and recipient as a hearing dog partnership. Each recipient is allocated a partnership instructor for ongoing partnership support in their local community. Initially, partnership instructors visit regularly to offer bespoke advice and support, tapering to a minimum annual visit once the partnership is established, although recipients can still contact them for support at any time and attend community activities, events, and workshops. Routine visits ensure the recipient is maintaining the dog’s welfare, standard of general behavior, and sound work skills training, thus providing an opportunity for the recipient to request advice and support on these issues or other areas of concern. A hearing dog partnership can last up to 10 years, after which the dog is retired.Once an application to Hearing Dogs for Deaf People is accepted, a client advisor liaises with the applicant over 3 to 6 months to create a detailed understanding and written Hearing Dogs for Deaf People report that the cost of training a dog and creating and supporting a partnership throughout the dog’s working life is around UK £40,000  .Although Hearing Dogs for Deaf People retain ownership of their hearing dogs and cover the costs described above, recipients are responsible for covering day-to-day costs such as food, routine veterinary care, and insurance.A literature review of evaluations of assistance dogs was conducted to make the case for this research . This rThe study will comprise a single-center, superiority randomized controlled trial (RCT) with nested economic and qualitative evaluations. In developing the protocol, the research team worked closely with Hearing Dogs for Deaf People to ensure the design fits with standard processes for creating hearing dog partnerships, the highly variable time it takes to match an applicant with a dog, and Hearing Dogs for Deaf People’s commitment to create a partnership within 3 years of application.The overall aim of the study is to evaluate the impact of a hearing dog partnership on the lives of individuals with severe or profound hearing loss.The study objectives are as follows:To determine the impact of a hearing dog partnership on mental well-being, as measured by the Short Warwick-Edinburgh Mental Well-Being Scale (SWEMWBS) , 6 monthTo determine the impact of a hearing dog partnership on secondary outcomes of impairment in functioning, anxiety, depression, and health-related and social care–related quality of life 6 months postreceipt of a hearing dog.To conduct a nested economic evaluation to investigate the cost-effectiveness of hearing dogs.To conduct a nested longitudinal qualitative study to describe the impacts of a hearing dog on recipients’ lives, and the mechanisms by which these impacts occur.To gather initial data on the long-term outcomes of having a hearing dog.The setting is Hearing Dogs for Deaf People, the only organization accredited to provide hearing dogs to UK residents. Research participants are first-time applicants for a hearing dog who may reside anywhere in the United Kingdom.The inclusion criterion is first-time applicants for a hearing dog, aged 18 years and older.The exclusion criteria include the following:Individuals aged 17 years or younger.Individuals requiring a dog who can provide sound and vision support.Individuals who are replacing a retiring hearing dog.Individuals with a learning disability (indicated by the use of a proxy during the application process).Individuals still awaiting a hearing dog but whose application is at a stage past the point where randomization could take place. These applicants will have the opportunity to complete the research materials and form a part of an exploratory longitudinal cohort study.The intervention includes the receipt of a hearing dog specifically matched and trained to support the needs of the applicant. The comparator includes not having a hearing dog. Practices regarding cessation of a hearing dog partnership should align with the standard Hearing Dogs for Deaf People protocols. Outside of the intervention, participants should receive care as usual.Recruitment to the trial took place between March 2017 and March 2018. Hearing Dogs for Deaf People will screen all applications during this period. Typically, the charity receives over 200 applications annually. For those fulfilling the study eligibility criteria, Hearing Dogs for Deaf People will post a study recruitment pack . Applicants wishing to participate can choose to complete the study materials on paper or on the Web in either English or British Sign Language (BSL) via Qualtrics, a Web-based survey platform. These will be returned directly to the research team. In the case of nonresponse, Hearing Dogs for Deaf People will send up to two reminders (text message and email or post). Where possible, reasons for decline will be obtained. Applicants not recruited to the study will follow standard Hearing Dogs for Deaf People procedures and timelines.Randomization will be conducted centrally by the York Trials Unit (YTU), using an allocation schedule generated in Stata v15. During their hearing dog assessment, study participants will be categorized by a senior practitioner within Hearing Dogs for Deaf People, with regard to their presenting needs, as follows:None: no remarkable or particular needs.Personal: predominantly personal needs, this might include particular health concerns or mobility.Environmental: predominantly environmental needs, this might include an inner city location or the presence of cats in the home.Both: significant personal and environmental needs.When the profile of 2 individuals with the same categorization of need is completed, they will form a pair and be randomized together, one to each group using block randomization with a block size of 2:Arm A: matching with a hearing dog occurs within usual timelines orArm B: matching accelerated, receive a hearing dog at least six months earlier than those in arm A.The allocation sequence will be generated by the trial statistician (CF) who has no involvement in the recruitment of participants. As pairwise randomization is being employed, ie, the randomization of 2 participants at a time, allocation is concealed.Participants will be blinded to their group allocation. Study team members who are actively involved in the administration of the trial will not be blinded.Follow-up questionnaires will be administered by the research team via post or email according to participants’ preferences. Postal, email, and text reminders and an incentive (£20 per data collection time point) will support retention to the study.The selection of outcome measures was informed by the existing literature and in consultation with Hearing Dogs for Deaf People and hearing dog recipients. All standardized measures are available in BSL, and these versions have satisfactory psychometric properties -29.none of the time to all of the time). Respondents indicate the response that best describes their experience over the last 2 weeks.The SWEMWBS  comprisetoday.The EuroQol 5 Dimensions (EQ-5D-5L) is a measure of health-related quality of life capturing five domains: mobility, self-care, usual activities, pain/discomfort, and anxiety/depression . Each donot at all to very severely).The Work and Social Adjustments Scale (WSAS) measures impairment in functioning and comprises five items, one related to work and the remainder to social functioning . Respondnot at all to nearly every day). Clinical cutoffs for BSL versions have been established [Two measures of mental health will be used: the Patient Health Questionnaire (PHQ-9) and the Generalized Anxiety Disorder Assessment (GAD-7) ,33. Respablished .Descriptive outcomes include the status of the hearing dog partnership (intact vs ceased); use of statutory, third sector, and private health and social care services in the past 4 weeks; and employment status.The Hearing Dog Questionnaire  will be not at all challenging to very challenging) and free-text response formats .Study participants who receive a hearing dog during the trial will also be asked about the benefits and challenges of their hearing dog partnership using fixed response  will opt to join the study. Allowing for 20% attrition at T1, this will result in a trial sample at T1 of 128. This pragmatic sample size will provide 80% power at 5% significance to detect an effect size of 0.5 of a standard deviation on our primary outcome measure, the SWEMWBS. This is a moderate-to-large effect size.Trial analyses on the effectiveness of hearing dogs will be conducted on an intention-to-treat basis, including all participants in the arm to which they were randomized. Analyses will be conducted in SPSS 23 or later, using 2-sided statistical tests at the 5% significance level. The flow of participants through each stage of the trial will be presented in a Consolidated Standards of Reporting Trials diagram. The primary analysis will estimate the difference in the SWEMWBS scores at T1 between arms A and B using linear regression, adjusting for the baseline measure and the individual’s presenting needs (identified before randomization). The difference between arms in the SWEMWBS score at T1 and corresponding 95% CI will be presented. Model assumptions will be checked, and if they are in doubt, data transformations, alternative distributions, or nonparametric alternatives will be considered. The secondary outcomes  will be analyzed in the same way.Sensitivity analyses will investigate the robustness of the findings given any nonadherence to the protocol. Application withdrawals, placement cessations, and adverse events will be summarized for each group.An exploratory analysis of outcomes at T2 will analyze the data using the same model as for the T1 with the caveat that there will be systematic missing data as T2 data are not collected from arm A participants who have received a hearing dog by this point. Further exploratory analyses will examine the longer-term outcomes collected for study participants where data collection at 18 (T3) and 24 (T4) months post receipt of a hearing dog is achieved. For these analyses, time will be added as a variable to the regression model.To date, the properties of the Hearing Dog Questionnaire have not been tested to determine whether the items perform as a scale. We will use classical test theory  to deterThe Statistical Analytical Plan will be uploaded to the trial registry.An economic evaluation will be conducted to determine whether hearing dogs are value for money. It will be undertaken from multiple perspectives to inform value for money considerations for different potential decision makers. Perspectives will include (1) a voluntary sector perspective (including costs to Hearing Dogs for Deaf People and of volunteers); (2) a social care perspective ; (3) a health care perspective ; (4) a public sector perspective ; and (5) a broader perspective considering costs to the voluntary sector, social care, health care, and costs incurred by the recipients of hearing dogs. The choice of outcome will be perspective dependent, choosing the most relevant outcome(s) for each decision maker, with social care quality-adjusted life years (SC-QALYs) and health quality-adjusted life years (H-QALYs) being key outcomes to consider. To determine cost-effectiveness, incremental costs and units of effect will be compared with relevant cost-effectiveness thresholds -38 usingData on costs and resource use will be collected from Hearing Dogs for Deaf People  and study participants at each data collection point . Costs will be calculated by applying unit costs to resource use. National unit costs   will be For outcomes, data on health-related quality of life will be collected using the EQ-5D-5L questionnaire at multiple time points. SC- and H-QALYs will be estimated using these data. H-QALYs will be calculated using the EQ-5D-5L score  and the A regression analysis will be undertaken to account for any baseline differences in study participants between the trial arms using appropriate techniques to account for non-normality of outcomes and costs data . Decisioactive ingredients of a hearing dog partnership, outcomes of partnerships , the processes by which changes in outcomes (or not) are perceived to occur, and views on the process by which partnerships are created and supported.The qualitative study seeks to understand the It will include a longitudinal study of 15 study participants who have received their hearing dog. This number provides pragmatic balance, allowing both for (1) recruitment to cover the range of variables in the purposive sampling frame (see below) and (2) the capacity and resource demands of the longitudinal approach. A purposive sampling frame will be used  to ensure a range of factors and circumstances are represented. Face-to-face interviews will take place approximately 4 and 10 months post receipt of a hearing dog. Participants will be able to participate in English or BSL. Depending on the language used, we will seek permission to audio or video record the interview. For interviews conducted in BSL, a detailed summary of the interview will be produced by the interviewer, an approach we have used successfully in the past . Intervimatch with a hearing dog, n=1 focus group); dog trainers , and partnership instructors . The focus group with client advisors will explore preparation of applicants for a hearing dog, applicants’ expectations and concerns, and factors affecting efficiency and quality of the matching process. Focus groups with trainers will explore dog and person characteristics, early indicators of a successful match and the active ingredients of the intervention. Focus groups with partnership instructors will explore factors that hinder or facilitate positive outcomes of hearing dog partnerships, the maintenance of the partnership, what constitute the active ingredients of the intervention, and experiences of interfacing or collaborating with statutory services. The number of focus groups per staff group reflects differences in size of each workforce. Focus groups for trainers and partnership instructors will take place in both of Hearing Dogs for Deaf People’s training bases (South and North England). Overall, the number and location of groups will allow the research team to collect data of sufficient breadth and depth.A cross-sectional qualitative study will explore the views and experiences of Hearing Dogs for Deaf People staff regarding factors that hinder or facilitate positive outcomes of the hearing dog partnership. Focus groups  will be used to gather views and experiences of the three groups of staff most involved with hearing dog applicants and recipients: Client advisors  comprising 10 individuals with a hearing dog has been formed. The panel will meet virtually using an online forum and group email. We will seek the views of the UAP on all aspects of the project, particularly the design and content of study information and consent materials, analysis of qualitative data, interpretation and synthesis of findings, and the dissemination strategy. Discussions with the UAP will be shared with the study steering committee. Hearing Dogs for Deaf People will also be consulted about dissemination and impact pathways, and we will advise them regarding their own dissemination of study findings.A study steering committee has been appointed comprising academics, researchers, and hearing dog recipients. The steering group will meet on four occasions over the course of the study.The study protocol (v3 12/02/2019) included the original application and subsequent amendments (as required) received a favorable ethical opinion from the University of York Department of Social Policy and Social Work Research Ethics Committee (SPSW/S/17/1).The study has been designed so that no participant will wait longer to receive a hearing dog than is usually expected. The nature of the two arms and the process of randomization will be made clear in study information materials as will the fact that participation is voluntary and choosing not to participate will not affect their application to Hearing Dogs for Deaf People or the service they receive. Study information materials will be developed in consultation with the user advisory panel.All participants will be required to provide written, informed consent on joining the study. Separate consent will be obtained for the nested qualitative study. Participants will be informed they can withdraw from the study at any time without this influencing their application.All study-related information will be stored securely at the University of York. Participant information will be stored in locked filing cabinets in areas with limited access. All outcomes data will be anonymized and given a coded ID. Records that contain names or other personal identifiers such as address and consent forms will be stored separately from outcomes data. Electronic data will be saved on a secure university filespace, access restricted to members of the research team that have a role analysis. Following the study, all data will be archived and stored in accordance with the University of York guidelines.All amendments to the protocol will be approved by the research ethics committee.A summary of the findings will be published by the National Institute of Health Research’s (NIHR) School for Social Care Research. Study findings will also be reported in open access, peer-reviewed journals and at relevant conferences. Participants will be sent a summary of the findings.Given the nature of the trial, the trial does not have a data monitoring committee.The study will record and report any details of serious adverse events.This trial does not have an audit procedure in place.The trial manager (LS) will oversee access to data. Cleaned data sets will be shared with the PI (BB), supervising statisticians (CF and CH), and economic team . Data dispersed to the research team will be blinded of any identifying participant information.Not applicable for this study.The study is funded by an NIHR School for Social Care grant, and recruitment commenced in March 2017. Recruitment is now complete, and 165 participants were randomized. Data collection is ongoing.This is the first time an RCT design has been used to evaluate the impacts of the hearing dog partnership. The findings from this study will provide, for the first time, strong and reliable evidence on the impact of having a hearing dog on people’s lives in terms of their quality of life, well-being, and mental health.In addition, we have shown that it is possible to do research that collects robust data on the impacts of assistance dogs on people’s lives. We think this study will encourage further research in this area, including for other types of assistance dogs.The study findings will be of relevance to people with hearing impairment, Hearing Dogs for Deaf People and its supporters, and statutory services responsible for the care and support of people with hearing impairments . Findings will support evidence-informed policy making, service development, and practice. Study findings will also be relevant to assistance dog organizations, in the United Kingdom and elsewhere.


Lactobacillus to replace Escherichia induced by H9N2 AIV, so as to resolve the intestinal flora disorder. In addition, baicalin can effectively prevent intestinal bacterial translocation of SPF chickens' post-H9N2 AIV infection, thus inhibiting secondary bacterial infection. Furthermore, baicalin can effectively treat H9N2 AIV-induced inflammation by inhibiting intestinal structural damage, inhibiting damage to ileal mucus layer construction and tight junctions, improving antioxidant capacity, affecting blood biochemical indexes, and inhibiting the production of inflammatory cytokines. Taken together, these results provide a new theoretical basis for clinical prevention and control of H9N2 AIV infection-induced secondary bacterial infection and inflammation.H9N2 subtype avian influenza virus (H9N2 AIV) is a low pathogenic virus that is widely prevalent all over the world. H9N2 AIV causes immunosuppression in the host and often leads to high rates of mortality due to secondary infection with Escherichia. Due to the drug resistance of bacteria, many antibiotics are not effective in the treatment of secondary bacterial infection. Therefore, the purpose of this study is to find effective nonantibiotic drugs for the treatment of H9N2 AIV infection-induced secondary bacterial infection and inflammation. This study proves, for the first time, that baicalin, a Chinese herbal medicine, can regulate  Orthomyxoviridae family [H9N2 avian influenza virus (AIV) is a kind of negative strand RNA virus that belongs to the influenza virus A genus of the e family . H9N2 ave family , 4. Althe family . H9N2 AIe family . H9N2 AIe family , 8.Escherichia coli [Escherichia coli) and other pathogens, resulting in high rates of mortality in poultry [The commensal gut microbiota has been reported to play a key role in viral pathogenesis by regulating the immune response against influenza virus . Howeverhia coli . Many cl poultry , 13. The poultry , 14, 15.Scutellaria baicalensis Georgi that is widely used as a traditional Chinese herbal medicine for its anti-inflammatory activities [Baicalin is a flavonoid compound purified from the medicinal plant tivities , 17. Baitivities . In additivities . Baicalitivities . HoweverThe use of animals in this study was approved by the Animal Care Committee of South China Agricultural University .Escherichia coli [The H9N2 subtype avian influenza strain A/Chicken/Henan/SH01/2015 SH0115) (GenBank No. KT023065) was preserved in our laboratory and was used in all related experiments that can cause high morbidity and mortality due to diarrhea and secondary bacterial infections such as 5  was dripped into nasal cavity of chickens in mock group and feeding baicalin group. Those 80 1-day-old SPF chickens were used for the following study: 16s rRNA high-throughput sequencing of intestinal microbiota, isolation of microflora from visceral organs, histological examination of intestinal segments and villus conditions, detection of antioxidant and blood biochemical indexes, and detection of intestinal immune-related cytokines, ileum mucus layer structure, and tight junction-related factors by quantitative real-time PCR (RT-qPCR).Among the 128 1-day-old SPF chickens, 80 1-day-old SPF chickens were randomly divided into 4 groups with 20 chickens per group: a mock group, an H9N2 infection group, a baicalin-fed group, and an H9N2 AIV infection plus baicalin-fed group. They were fed in a negative pressure isolator. The specific experimental groups are shown in 6 TCID50/0.1 mL, which lasted for three days. On the third day after virus infection, all chickens were fed with Neongreen bacteria at a dose of 300 μL 1 × 109 CFU/mL per chicken.The other 48 1-day-old SPF chickens were used for the study of Neongreen-tagged bacteria isolation. Those 48 1-day-old healthy SPF chickens were divided into four groups: a mock group, an H9N2 AIV infection group, a baicalin group, and an H9N2 AIV infection with baicalin-feeding group. Baicalin was introduced to the corresponding groups at the age of 1 day. The additive amount of baicalin was 0.5 g baicalin powder per 1000 g feed. At the age of 9 days, the infection dose of H9N2 virus was 3-fold 10Three chickens were randomly selected from the mock group, H9N2 infection group, baicalin-fed group, and H9N2 AIV infection plus baicalin-fed groups, respectively, at 5 days and 12 days postinfection (dpi) with H9N2 AIV. After carotid artery bloodletting, the contents of the ileum were taken and placed in an aseptic freezer under aseptic conditions. Using the FastDNA SPIN Kit for Soil kit, the total DNA of the ileal contents of the chicken was extracted according to the steps described in the manufacturer, and the purity, concentration, and integrity of the extracted DNA were detected by NanoDrop 2000 spectrophotometer  using the 260/280 nm absorbance ratio and 1% agarose gel electrophoresis. In order to ensure the accuracy and reliability of the follow-up data analysis, the V3–V4 variable regions of 16s rRNA of the qualified samples were amplified by PCR. After the Illumina Miseq PE300 sequencing was completed in the sequencing platform, the effective sequences were filtered and optimized, and OTUs  were defined to analyze the biological information of all sequences according to the similarity of more than 97% sequences.μL homogenate in each tube was evenly applied on the prepared LB plate. After the treated plates were cultured in a constant temperature incubator at 37°C for 12 hours, the growth of bacteria on each plate was recorded statistically, and those with colonies were recorded as positive, and those without colonies were negative.Three chickens were randomly selected from the mock group, H9N2 infection group, baicalin-fed group, and H9N2 AIV infection plus baicalin-fed group, respectively, at 5 days and 12 days postinfection (dpi) with H9N2 AIV. After carotid artery bloodletting, livers, lungs, and mesentery were obtained under aseptic conditions. An appropriate amount of corresponding tissue was put into the grinding tube with 1 mL normal saline. After each tissue was fully homogenized, the mixture of 200 E. coli bacteria modified by the NeonGreen fluorescence labeling gene. Aseptic collection of a mixture of the inner wall and lumen of the ileum, liver, lungs, and mesentery was conducted. The homogenate was homogenized in a sterile homogenate tube containing 1 mL saline. One hundred microliters of homogenate mixture was taken to the LB plate with ampicillin resistance, and the bacterial solution was coated and cultured in a 37°C incubator for 12 hours. When bacterial growth on the plate was observed, the colony growth was judged to be positive, while noncolony growth was judged to be negative.Slaughtering samples were taken at 12, 24, 36, and 48 hours after the administration of Neongreen bacteria in the mock group, H9N2 infection group, baicalin-fed group, and H9N2 AIV infection plus baicalin-fed group, respectively. Neongreen-tagged bacteria was the Neongreen Three chickens were randomly selected for the mock group, H9N2 infection group, baicalin-fed group, and H9N2 AIV infection plus baicalin-fed group, respectively, at 5 days and 12 days postinfection (dpi) with H9N2 AIV. After carotid artery bloodletting, the ileum and cecum were fixed in formalin and made into HE staining sections. Intestinal sections were observed with a Nikon Eclipse ci optical microscope and analyzed and photographed with the NIS-Elements F 4.00.00 software . Three intact intestinal villi were selected for each section. The height and corresponding crypt depth of selected intestinal villi were measured, the villus height to crypt depth ratio was calculated, and the average value was taken as the final measured value.Three chickens were randomly selected from the mock group, H9N2 infection group, baicalin-fed group, and H9N2 AIV infection plus baicalin-fed group, respectively, at 5 days and 12 days postinfection (dpi) with H9N2 AIV. Two milliliters of blood was collected from the aseptic vein under the wing. The blood was placed at room temperature without anticoagulant treatment, and the serum was isolated by 2143 g centrifugation for 10 minutes after agglutination. The kits used for the determination of total antioxidant capacity, glutathione peroxidase, superoxide dismutase, and malondialdehyde were purchased from the Nanjing Jiancheng company . The procedures were performed according to the instructions in the different kits. Serum urea nitrogen, low-density cholesterol, high-density cholesterol, aspartate aminotransferase, total cholesterol, and alanine aminotransferase concentrations were measured by a Mindray BS-5800M automatic biochemical analyzer .μg RNA of each sample was reverse-transcribed into cDNA using the RecerTra Ace qPCR RT Master Mix with gDNA remover , and the cDNA was stored at -20°C. GAPDH was as the internal reference gene. The target gene was amplified by RT-qPCR, and the relative expression of each gene was calculated by 2-△△Ct [Three chickens were randomly selected from the mock group, H9N2 infection group, baicalin-fed group, and H9N2 AIV infection plus baicalin-fed group, respectively, at 5 days and 12 days postinfection (dpi) with H9N2 AIV. Under an aseptic environment, a segment of the ileum was cut off and put into a 2 mL grinding tube containing 1 mL RNAiso. According to the sequences published in GenBank, primers for RT-qPCR detection of intestinal inflammation-related cytokines were designed and the related primer sequences are shown in y 2-△△Ct .t-tests, and then one-way analysis of variance (ANOVA) with the Tukey post hoc tests was used to compare the differences among different treatment groups. For each separate set of experiments, three independent biological replicates were evaluated. Six technical replicates were used for RT-qPCR. Data are presented as the mean ± standarddeviation.Statistical analysis was performed using the SPSS 13.0 statistical software . The relative abundance analysis of microflora, intestinal structural damage analysis, ileal tight junction protein, antioxidant capacity analysis, blood biochemical indexes, and inflammatory cytokine analysis were analyzed by Student's Lactobacillaceae at 5 dpi significantly decreased in the H9N2 AIV infection group, while the quantity of Enterobacteriaceae significantly increased at the family level  groups was analyzed by 16S rRNA high-throughput sequencing. Firmicutes were the major bacterial phyla present in all samples from the different groups at 5 days postinfection (dpi) . The proly level . Furtherus level . These r Figures . The resIn addition, compared with the control group at 5 dpi, the number of OTUs increased and the number of specific OTUs increased by 214 and 197 in the baicalin-added group. The number of OTUs increased and the number of specific OTUs decreased by 190 and 153, respectively, in the H9N2 AIV infection group. Compared with the H9N2 AIV infection group, the number of OTUs and the number of specific OTUs in the H9N2 AIV infection with baicalin group increased by 165 and 154, respectively . The resProteobacteria and Escherichia caused by H9N2 AIV infection and restore the concentrations of Firmicutes and Lactobacillus in the intestine at the phylum and genus levels, respectively, at 12 dpi     (P < 0.01) (P < 0.01)  . The exp < 0.01) . These r Figures . These rP < 0.01 and P < 0.05)   . These rP < 0.01)   (P < 0.01)  . Besides Figures . Those rIFN-r, TNF-α, IL-22, IL-17A, IL-6, and IL-1β are important proinflammatory cytokines. The purpose of this study was to investigate whether H9N2 AIV infection promotes changes in the above proinflammatory factors in intestinal epithelial cells, and then to explore whether baicalin can inhibit changes to proinflammatory factors caused by H9N2 AIV. As shown in Figures IFN-γ, TNF-α, IL-22, IL-17A, IL-6, and IL-1β were significantly upregulated in the infected group at 5 dpi and 12 dpi compared with mock-infected chickens. Furthermore, the addition of baicalin to the infected group significantly decreased the mRNA expression of IFN-γ, TNF-α, IL-22, IL-17A, IL-6, and IL-1β (P < 0.01). These results demonstrated that the addition of baicalin could effectively inhibit inflammation of the chicken intestine caused by H9N2 AIV infection.Inflammation in body tissues is accompanied by changes in proinflammatory cytokines . IFN-r, H9N2 AIV is widely distributed, and the harm it causes is long-lasting and difficult to control. In recent years, secondary bacterial infections caused by H9N2 AIV have been reported from time to time , 13, 24.Escherichia coli, in the intestinal tract is low. When intestinal homeostasis is broken, for example, due to viral infection, the proliferation of conditional pathogenic bacteria is promoted, causing intestinal disorders. In this study, we confirmed that H9N2 AIV infection significantly promotes the proliferation of endogenous Enterobacteriaceae, which is consistent with our previous results [Escherichia, restore the microecological environment destroyed by H9N2 AIV, and restore Lactobacillus as the dominant flora at 5 and 12 dpi, illustrating that baicalin can effectively inhibit the increase in Escherichia caused by H9N2 AIV infection and restore the number of beneficial bacteria in the intestinal tract. In the intestinal cavity, bacteria and viruses can penetrate the intestinal mucosa and enter other tissues, organs, and circulatory systems, resulting in bacterial translocation, leading to disease [The intestinal microecological flora of poultry plays an indispensable role in disease control and immune regulation. According to the relationship between the intestinal tract and host, intestinal microbes can be divided into intestinal symbiotic bacteria, conditional pathogenic bacteria, and pathogenic bacteria. The concentration of conditioned pathogens, mainly facultative anaerobes such as  results . Further disease . We confThe main pathological manifestations of H9N2 infection are enlargement of the intestinal lymph nodes and necrosis of epithelial cells in the ileum and cecum , 29. TheOxidative stress is involved in several acute and chronic pathological processes, such as acute and chronic kidney disease and neurodegenerative diseases . In addiIL-17A, IL-22, IL-6, IL-1β, IFN-γ, and TNF-α increased at 5 and 12 days post H9N2 AIV infection, and these results are consistent with our previous research results and other reports [Blood has the function of maintaining the stability of the environment in the body, and the serum biochemical index reflects the physiological state and health status of the animal body, which can be used as a standard to measure the health of animals . Increas reports , 46. FurEscherichia by intestinal beneficial bacteria. In addition, baicalin can inhibit the translocation of intestinal bacteria caused by H9N2 AIV infection, thereby inhibiting secondary infection. Furthermore, baicalin has the effect of minimizing intestinal structure injury, inhibiting damage to ileal mucus layer construction and tight junctions as well as injury to SPF chickens' post-H9N2 AIV infection by affecting blood biochemical indexes, improving the antioxidant capacity, and inhibiting inflammatory cytokines, alleviating the inflammatory response of SPF chickens caused by H9N2 AIV infection. This study provides a new theoretical basis for clinical prevention and control of secondary bacterial infection and inflammation caused by H9N2 AIV infection.This study proves, for the first time, that baicalin can effectively inhibit the intestinal microbial disorder caused by H9N2 AIV infection and promote the replacement of


This study aimed to investigate the possibility of using lifetime liveweight and liveweight change and previous body condition scores to predict current body condition scores in Romney ewes. Models using a ewe’s lifetime liveweight record alone were poor at predicting ewe body condition scores. A combination of lifetime liveweight, liveweight change, and previous body condition scores improved body condition score prediction. If higher accuracy can be achieved, these prediction equations can be incorporated into the electronic weigh heads of modern weigh systems to automatically give farmers predictions of the body condition score (BCS) of an individual during routine weighing. This would benefit farmers by allowing for targeted nutritional management of individual animals to maximize overall flock productivity.Ovis aries) is a widely used subjective measure of body condition. Body condition score and liveweight have been reported to be statistically and often linearly related in ewes. Therefore, it was hypothesized that current BCS could be accurately and indirectly predicted using a ewe’s lifetime liveweight, liveweight change, and previous BCS record. Ewes born between 2011 and 2012  were followed from 8 months to approximately 67 months of age in New Zealand. Individual ewe data was collected on liveweight and body condition scores at each stage of the annual cycle . Linear regression models were fitted to predict BCS at a given ewe age and stage of the annual cycle using a ewe’s lifetime liveweight records . Further, linear models were then fitted using previous BCS and changes in liveweight, in addition to the lifetime liveweight records (combined models). Using the combined models improved (p < 0.01) the R2 value by 39.8% (from 0.32 to 0.45) and lowered the average prediction error by 10% to 12% (from 0.29 to 0.26 body condition scores). However, a significant portion of the variability in BCS remained unaccounted for (39% to 89%) even in the combined models. The procedures found in this study, therefore, may overestimate or underestimate measures by 0.23 to 0.32 BCS, which could substantially change the status of the ewe, leading to incorrect management decisions. However, the findings do still suggest that there is potential for predicting ewe BCS from liveweight using linear regression if the key variables affecting the relationship between BCS and liveweight are accounted for.The body condition score (BCS) in sheep ( Ovis aries) is a widely used subjective measure of the degree of body fatness [The body condition score (BCS) in sheep  to better manage flocks, farmers do not regularly use this technique. For example, while 96% of Australian producers indicated they monitored the body condition, only 7% conducted hands-on BCS  used BCSThe relationship between BCS and LW is documented in sheep ,10, withIn large extensive flock systems, farmers regularly weigh sheep and are increasingly using electronic tags . Both coThe current study utilized data collected between 2011 and 2016 from two commercial New Zealand sheep farms (A and B) as part of normal routine farm management. All ewes (Romney breed) were weighed (to the nearest 0.1 kg) using static digital weighing scales . Body condition scores were undertaken by experienced assessors using a 1–5 scale  with sheep assessed to the nearest 0.5 of a BCS ,5 at fouData were analyzed using R program version 3.3.4  with pacThe body condition score data is both discrete and ordered in nature, which makes multi-class classification regression approaches such as ordinal logistic or nominal regression more suitable for its analysis. However, when the underlying assumptions are grossly violated or when classes are extremely imbalanced , classifInitially, the best predictor combinations for each BCS were selected through the regularization and variable selection technique implemented in the R program  using thAll models were constructed, fitted, and validated using algorithms, implemented in four steps. The steps included; (i) data partitioning, (ii) resampling, (iii) model training, and (iv) validation. Data partitioning involved dividing the initial dataset (with stratification preserving the class proportions) into training and testing datasets in a ratio of 3:1, with replacement. Resampling involved using bootstrapping and aggregation  procedurp < 0.05). Using the selected best predictors for each BCS, LM regression equations were fitted to predict the current BCS using lifetime (present and previous) liveweight records . Later, the models were modified by using previous liveweight change and BCSs in addition to lifetime liveweight (combined models). Consequently, forty-eight (48) regression equations were generated for BCS prediction, half of which were from using liveweight alone models and the remaining half from the combined models. Lifetime measurements refer to those ewe measurements taken at the same and previous time points, whereas previous measurements only refer to those preceding the current one. Liveweight change was defined as sequential retrospective change in liveweight between individual time points.Two multiple regression approaches were evaluated for the possibility of predicting BCS on a full scale, namely, the general linear model (LM) using the generalized least squares (GLS) and linear mixed-effects model (LMM) procedure in the nlme package . The LM 2: accounting for number of predictors) and the root mean square error (RMSE). The validation for each BCS prediction model was conducted using the testing dataset, each repeated 1000 times. Several metrics were considered when assessing the quality of models, including the coefficient of determination (R2 or unadjusted R2), bias, root mean squared error (RMSE), residual prediction deviation (RPD), and the ratio of performance to interquartile distance (RPIQ) [2, RPD, and RPIQ, and the lowest RMSE, MAPE and RPE. In addition, RPD was classified [The calibration model performance (based on training dataset) was assessed using two metrics ,29: The e (RPIQ) . The sucassified  into thrassified , RPIQ waThere was an association between liveweight and BCS in all age groups and stages of the annual cycle, but the association was characterized as being weak to moderate a,b. The 2 (based on training dataset) value showed no clear pattern  were included in linear regression equations. Across age groups, the change in adjusted R pattern . The adjThe average number of liveweight predictors (significant variables) for BCS prediction was seven (1 to 16), with no clear pattern of change over time . To impr2 values  or 1.4 times, and the number of significant predictors at each stage of the annual cycle by 3.6  times. A significant portion of the variability in BCS remained unaccounted for (38–89%) in the combined models, with some of the initial liveweight variables in the liveweight alone models being considered non-significant (p > 0.05) in the combined models.The adjusted R2 values  for ewe The BCS model prediction error metrics  varied across age group and stage of the annual production cycle when liveweight or combined models were used to predict BCS . The avep < 0.01) reduced compared to the liveweight alone models. The average prediction error associated with BCS prediction using the combined models in terms of MAE and RMSE was reduced by 0.04 (10% to 12%) body condition scores. Overall the combined models improved BCS prediction from weak to reasonable (PRD: 1.40) or good (RPID: 1.75). The results also showed positive and negative biases for both models, and an indication of the tendency to underestimate or overestimate BCS measurement.The model prediction error metrics for the combined models varied across age group and stage of the annual production cycle but were significantly  remained unaccounted for in both liveweight alone and combined models, respectively. Potential reasons for the apparent failure for both liveweight alone and combined models to account for more of the variability in BCS include BCS binning (due to not being a continuous variable), assessor consistency over time, losses in liveweight due to gut-fill and urination when ewes are weighed at different times, fleece weight and wetness, and confounding of liveweight with conceptus weight. The consistency of the BCS data can vary between 5% to 27% and 40% to 60%, and within 16% to 44% and 60% to 90% for inexperienced and experienced assessors, respectively [The variability in BCS explained for both liveweight and combined models increased with the number of predictors in the model. This was expected as it is known that as the number of predictors that significantly relate to the dependent variable increase, the proportion of the variance due to the regression increases . Howeverectively . Liveweiectively ,36. Thusectively , which cectively . Therefoectively .In the present study, the prediction models using liveweight alone had large error (MAE and RMSE) and low RPD and RPIQ values, which led to high error rates. Combined models reduced the magnitude of all the prediction error metrics to near acceptable levels. Although an error (MAPE or RPE) up to 20% is acceptable for setting dosage rates in the veterinary pharmaceutical industry , an erroThe combined models improved the proportion of variability in BCS accounted for, as well as the accuracy metrics across age groups and stages of the annual cycle and over time (years), compared to the liveweight alone models. This indicates that BCS could be better predicted if additional variables  were included in the multiple regression equation, rather than lifetime liveweight alone. These relationships could potentially be incorporated in electronic weighing systems that utilize lifetime data. This would be especially useful when applied to large extensively run sheep flocks. However, a significant portion of the variability in BCS remained unaccounted for (39% to 89%) even in the combined models. It is possible that the prediction models could be improved if additional information such as stage of pregnancy, number of fetuses carried, and time off feed was utilized and warrants further investigation.


To revisit the concept of retinectomy and the theory of mechanical forces on the retina occurring in rhegmatogenous retinal detachment (RRD) and to describe the potential application of radial retinectomy in RRD with advanced proliferative vitreoretinopathy (PVR).A literature search was performed to identify all English language articles reporting the use of retinectomy for the management of RRD with PVR. We reviewed the theoretical background of mechanical forces occurring in RRD.Detachment of the retina from the retinal pigment epithelium (RPE)/choroid is influenced by disequilibrium of several physical forces: tangential forces on the epiretinal membrane Relaxing radial retinectomy may potentially be applied in RRD with advanced PVR but has rarely been reported to date. Future studies are needed to evaluate its outcomes and long-term complications. Rhegmatogenous retinal detachment (RRD) is the most common form of retinal detachment  and is cTheoretically, in retinal detachment, mechanical forces caused by PVR play a key role in generating traction and ultimately reduce the success rate of retinal reattachment surgery . TherefoIn this review, we aimed to revisit the concept of retinectomy and the theory of mechanical forces on the retina during retinal detachment and to propose radial retinectomy as a potential approach specifically in the context of retinal detachment with advanced PVR.RRD is the most common form of retinal detachment. RRD occurs in association with a full-thickness retinal break or tear . The outPVR has attracted substantial attention over the last two decades for many VR surgeons because it may determine the complexity of the surgery and may also significantly influence the success rate of RRD repair . PVR is Mechanical stress at the site of RRD stimulates glial cells to release proliferation factors, causing macrophage and fibroblast proliferation and migration into the vitreous at this site , 18. RetThe ultimate goal of RRD surgery is to restore the retina to its original position to preserve optimal macular function and to prevent subsequent redetachment as a consequence of repeated proliferation . In the The concept of retinotomy was first described in 1979 by Machemer, who pioneered this method to manage the treatment of eyes with retinal incarceration due to traumatic scleral wounds . ŽivojnoDetachment of the retina from the RPE/choroid is influenced by several physical forces: retinal tensile force, retinal adhesive force, and intraocular fluid flow , 24. FigIn normal eyes Fig. a, the el surface –28. In ae retina , 30. Thimentclass2pt{minimmentclass2pt{minimmentclass2pt{minimIn principle, all approaches performed during retinal detachment surgery  aim to reduce the radial traction ument}FA . The natument}FA . Intraocument}FA . Howevere retina , 34. Thee retina . While ce retina , 33, 36.entclass1pt{minimaEarly surgical steps in closed posterior vitrectomy, including wound construction, vitrectomy, sub-retinal fluid drainage and removal of any existing epiretinal membrane, are generally routine procedures. Thorough retinal observation should be performed to determine the locations of retinal breaks and PVR extension before vitreous removal. In the presence of an epiretinal membrane or scar tissue, peeling or removal of the scar tissue causing retinal wrinkling is preferrably performed using forceps to lift the membranous edge. Frequently, complete retinal reattachment can be achieved after removal of scar tissue on the retinal surface without the need for retinectomy. However, in situations where retinal reattachment cannot be achieved and retinectomy is required, it is very important to carefully determine the sites for retinectomy in order to maximize preservation of the optic nerve and macula as much as possible. Anatomically, the density of photoreceptors peaks at the posterior pole, which is approximately 4.5–6 mm around the fovea centralis –39. TherCertain situations demand careful attention. First, in the presence of extensive cellular proliferation at the vitreous base, meticulous vitrectomy and membrane peeling must be performed. Radial retinectomy should be executed in the most heavily wrinkled area of the retina, because this area has the greatest tangential traction. Second, it is also important to note that in the case of thick circumferential membrane proliferation at vitreous base with strong adhesion to the retina, the peeling technique alone may not adequately remove the membrane and eliminate traction. This situation may require a radial incision in the retina across the circumferential membrane formation to relax the traction. These aforementioned techniques would induce relaxation of the retina and flatten the retina during fluid-air exchange, especially in the peripheral retina, anatomically the thinnest point. Importantly, neither of these techniques induce retinal dialysis or avulsion. Third, when further retinal thickening and shortening is also present (grade D PVR), combination of these techniques with an external encircling buckle or circumferential retinectomy can be considered to allow sufficient elimination of traction. However, a detailed technique combining radial and circumferential retinectomy is beyond the scope of this review, and thus will not be discussed.Relaxing radial retinectomy is performed considering the size or grade of the PVR and should target the area of fibrotic tissue or the site of thick PVR. Retinectomy can be applied one or two times depending on the extent of the PVR. In grade C1 PVR, radial relaxing retinectomy can be performed in 1 quadrant, whereas grade C2 or C3 PVR may require two or three quadrants  at the time of his first visit was 1/300 with an intraocular pressure (IOP) of 8 mmHg. The patient had no signs or history of uveitis, posterior abnormalities or diabetic retinopathy with or without posterior laser treatment. General eye examination showed normal anterior segment. Posterior segment examination showed subtotal retinal detachment in all quadrants involving the central macula, with full-thickness fixed retinal wrinkle between the 11 to 8 o’clock positions, mature and immature membranes, and subretinal fibrosis extending peripherally between the 4 and 8 o’clock positions at the vitreous base. We performed vitrectomy, membrane peeling and subretinal fibrosis removal. We could not entirely remove subretinal fibrosis due to its very strong adhesion to the retina and the retina could not be flattened during fluid-air exchange; therefore, we performed radial retinectomy at three sites: one at the 1 o’clock position superior and two at the 5 and 8 o’clock inferior across the remaining region of subretinal fibrosis (Fig. As outlined in Table 1, all previously published studies reported a considerable proportion of complications within some period after RRD surgery with retinotomy/retinectomy. During retinotomy/retinectomy, the entire retina, excluding its RPE, is removed. Direct exposure of intravitreal fluid to retinal pigment epithelial cells, which are responsible for controlling the osmotic gradient of fluid transfer, increases the rate of uveoscleral outflow, resulting in ocular hypotony . The posWhile relaxing radial retinectomy is less aggressive than circumferential retinectomy and has a smaller incised area of the retina, the potential complications that may occur following this particular technique remain less certain. Therefore, caution regarding all possible complications after retinectomy is very important, and surgeons should consider performing retinectomy as the last available option after all approaches have proven unsuccessful in flattening the retina to its original position.Treatment of RRD with PVR may be considered routine among VR surgeons, yet surgical management of this condition remains challenging because postoperative anatomical results can be unsatisfactory. Circumferential retinotomy/retinectomy, introduced few decades ago, has been widely used as one of important surgical approaches for RRD surgery with complex PVR and, more importantly, has greatly improved both clinical and most notably the anatomical outcome of the surgery, despite the potential occurrence of several complications. Relaxing radial retinectomy is another retinotomy/retinectomy technique that has not been widely applied or at least has been seldom reported to date, despite its strong theoretical foundation for application in RRD with severe PVR. Whether this technique can be a substitute for previous circumferential retinectomy approaches remains to be confirmed. Future studies are needed to determine the applicability and repeatability of relaxing radial retinectomy among VR surgeons and to evaluate its outcomes in various cases and possibly its comparability with previous circumferential retinectomy. Finally, regardless of whether relaxing radial retinectomy is superior or inferior to previously established retinectomy techniques, it may offer a good addition to a surgoen’s library, which eventually enrich the treatment options for different cases, after careful consideration of potential risks and benefits.A literature search of English language articles was performed in PubMed, Google Scholar or Scopus using the following keywords alone or in combinations: “rhegmatogeous retinal detachment”, “proliferative vitreoretinopathy”, “retinotomy”, “retinectomy”, “biomechanics”, “mechanics”, “pathophysiology”, “pathology”, “risk factors” and “treatment”. Manual search from list of bibliography of major articles was also performed to identify key references in this area. Articles were sorted and rated according to relevance and summarized to produce this review.