Telocytes (TCs) are a distinct stromal cell type described in many organs. The present study investigated the existence of TCs within the efferent ductules in camel and the changes that occur in their morphology and activity during active and inactive reproductive seasons. TCs in the camel had a cell body and multiple telopodes (TPs), and most TCs had indented nuclei that exhibited prominent intranucleolar chromatin. TCs exhibited seasonal differences which were evaluated by histochemistry, immunohistochemistry (IHC), Transimition electron microscopy (TEM) and scanning electron microscopy (SEM). The presence of TCs in camel efferent ductules has been confirmed by CD34 positive immunostaing. In addition to the expression of the vascular endothelial growth factor (VEGF) which was stronger in the summer season. TCs exhibited stronger immunoreactivity for progesterone and oestrogen alpha receptors (ESR1) in the spring than in the summer. In addition, TCs showed strong positive immunostaining for both vimentin and androgen receptor (AR). Several ultrastructural changes were observed in TCs during the two seasons. TPs in the summer season had delicate ramifications whereas, in the spring, TPs displayed fine arborization and became more corrugated. TCs acquired signs of exaggerated secretory activities in the spring; TPs became expanded and packed with secretory vesicles. Thus, we conclude that, hormonal alterations during the reproductive cycle impact the morphology and secretory behavior of TCs. They establish a cellular communication system and play a central role in the functional regulation of different types of cells and structures5. TCs have unique morphological features that distinguish them from other stromal cells. TCs possess cell prolongations; telopodes (TPs) which extend from the cell body. TPs form an interstitial labyrinthine network to allow cellular communication. TPs have thin segments or podomeres and interval expansions or podoms which are rich in mitochondria, endoplasmic reticulum, and caveolae6.Telocytes (TCs) represent a distinct type10 and are involved in mechanoreception11. TCs also have excitatory and inhibitory neurotransmitter receptors12. The role of these cells in organ regeneration has been studied in several organs, including the heart, lung, skeletal muscle, skin, meninges and choroid plexus, eye, liver, uterus, and urinary system13. Based on gene expression analyses, various functions have been suggested for TCs such as cellular signalling15, cell expansion and movement14, tissue homoeostasis, remodelling15 and repair13, embryogenesis16, morphogenesis17, angiogenesis15, suppression of oxidative stress and cellular ageing18, and protection against inflammation and oncogenesis19.Several broad communication functions have been described for TCs. TCs transmit nerve impulses to smooth muscle cells20. The types of cell contacts in TCs including direct apposition of the cell membranes of adjacent TCs, adherence , and gap junction. Gap junctions play a significant role in signal transduction between cells21. The paracrine functions of TCs depend on molecular transfer through extracellular vesicles; exosomes, ectosomes and multivesicular vesicles23.Cellular connections are prominent feature of TCs that provide functional support to other cells. Two modes of communication are described for TCs; cell contact or through paracrine signaling. Cell contact is classified according to cell type into homocellular or heterocellular contact. TCs exhibit various forms of heterocellular contacts, including minute junctions such as point contacts, nanocontacts, planar contacts and cell contacts with an intermembrane distance, allowing macromolecules to interact24. In the present study, we investigated TCs changes in response to hormonal changes in the genital organs in one of the seasonal breeder animals (camel) during the active and inactive reproductive seasons. We used samples of the efferent ductules for the one-humped camel obtained in two different seasons, summer and spring. Efferent ductules are important for sperm transportation and fluid reabsorption25. Camel breeding activity reaches maximum levels during the active or rutting period; in the winter and spring seasons while the breeding activity declines during the inactive or non-rutting period; summer and autumn27. The main goal of the present investigation was to recognize TCs in the efferent ductules, their distribution, identify camel TCs characteristics, their relations to other cells, and explore the activity of TCs during inactive and active seasons of the reproductive cycle.Microenvironment- dependent changes in TCs behavior have been previously described; Hormonal administration affects the morphology and activities of TCsThe present study was performed on efferent ductules collected from 10 clinically healthy mature camels (Camelus dromedarius) during the spring season (March-April) and during the summer season, which represents the inactive period (July-August). The materials were collected from the Bani-Adie slaughter house in Assiut-Egypt.Efferent ductules were dissected and were fixed using (a) Bouin’s solution for light-microscopic examination and (b) Karnovsky fixative  for semithin sections and electron microscopy.Samples fixed in Bouin’s solution were extensively washed in 70% ethanol (3 times for 24 h) to remove the fixative prior to subsequent tissue processing steps for paraffin block preparation; Fixed samples were dehydrated in ascending grades of alcohols at 70%, 80%, 90% and 100% for 90 min at each concentration. The samples were cleared using methyl benzoate. Dehydrated samples were then impregnated and embedded in Paraplast . Serial sections of 3–5 μm were cut using a Richert Leica RM 2125 Microtome, Germany and mounted on glass slides. Sections were kept in an incubator at 40 °C.et al.28 with modification. Stock solution: 50 mg acridine orange is dissolved in 10 ml of distilled water and stored in the refrigerator, . The pH of the staining solution was approximately 3 and the AO concentration was 0.01%.The procedure was according to that of Hoff, The staining procedure: 5-μm paraffin sections were dewaxed (2 times for 30 min) and rehydrated in a descending series of ethanol  and DW. Dried sections of glass slides were fixed with methanol and dried in a trough with AO staining solution (0.01 per cent). After 2 minutes of staining, the slides are washed gently with DW, dried and then analyzed using a Leitz DM 2500 microscope with the external fluorescent unit Leica EL 6000.29 at 4 °C overnight. Semithin sections (1 μm) were cut using an ultramicrotome Ultracut E  and stained with toluidine blue30.Specimens from efferent ductules were used for semithin sections. Small pieces 2.0–3.0 mm in length were fixed in Karnovsky fixative31 according to the following protocol: paraffin-embedded tissue sections (5 μm) were dewaxed, rehydrated, and rinsed 3 times in PBS (pH 7.4) for 5 min. Endogenous peroxidase was inhibited by soaking the sections in 1% H2O2 for 10 min at room temperature, followed by intense washing under running tap water for an additional 10 min. For antigen retrieval, the slides were heated in water bath (for 20 min) in citrate buffer (pH 6.0) using a water bath followed by cooling for 20 min  was carried out in 0.1 M sodium citrate buffer solution (pH = 6) for 4 min using a microwave (600 Watt). The sections were incubated at room temperature for 2 h with blocking solution , 1% bovine serum albumin (Roth) and 0.3% Triton X-100 (Fluka Bio Chemika) to minimize non-specific labelling and increase the permeability to the efferent ductule tissue. Sections were incubated 24 h at 4 °C with primary antibody against vimentin  and androgen receptor marker . To visualize the primary antibody the sections were first washed with PBS (3 × 10 min each), followed by incubation 2hrs (in dark) with the same blocking solution containing the secondary antibody conjugated to Alexa-594 . Following a 1x PBS wash, sections were incubated for 10 min with DAPI  to visualize the nuclei, and FITC-coupled Tomatolectin  to visualize the lectins (carbohydrate-binding proteins). The sections were washed again with PBS (3 × 10 min each). Finally, the sections were cover slipped with fluoro mount mounting medium , and were kept in the dark to preserve fluorescence until examination with an AxioImager M2 fluorescent microscope (Zeiss) and pictures captured with AxioCam HRc camera.Representative specimens from the efferent ductules were washed several times in normal saline and then fixed in a mixture of 2.5% paraformaldehyde and 5% glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.3, at 4 °C for 24 h. Thereafter the specimens were washed 4 times for 5 min in the fixation buffer and post fixed in 1% osmic acid in 0.1 M sodium phosphate buffer for additional 2 h at room temperature, followed by washing 4 times with 0.1 M sodium phosphate buffer for 15 min. The samples were dehydrated using different concentrations of alcohol; 50, 70, and 90% for 30 min at each concentration and 100% for 2 days  followed by isoamyl acetate for 2 days. The dehydrated samples were subjected to critical-point drying with a Polaron apparatus. Finally, the samples were coated with gold using JEOL- 1100 E ion sputtering device and observed with a JEOL scanning electron microscope (JSM 5400 LV) at 10 KV.Ultrathin sections were obtained by a Reichert ultra-microtome. The sections (70 nm) were stained with uranyl acetate and lead citrate and examined by a JEOL100CX II transmission electron microscope in the electron microscopy unit of Assiut UniversityScanned and transmission images were colored using the Photo Filter 6.3.2 program. To increase the visual contrast between several structures on the same electron micrograph.32. This has been done by the following steps: open image with CMEIAS Color Segmentation, then select “Process” from the menu items, and subsequently choose “Negative image34”.Negative images were performed using CMEIAS Color Segmentation, an improved computing technology used to process color images by segmenting foreground object of interest from the backgroundThe present study aimed to identify TCs in the camel efferent ductules and investigate the morphological changes of TCs and their secretory behavior in response to seasonal variation.TCs were identified in camel efferent ductules and, these cells were composed of the a cell body and TPs. TCs in camel efferent ductules were organized under the epithelium Figs  and 2A, The typical morphological features of TCs were identified for the first time using “Acridine Orange staining technique”. As observed in Fig. 1). Notably, ESR1 positive network was more complex in the spring than in the summer  that influence stem cells by paracrine or juxtracrine mechanisms56. Moreover, TCs established contact with blood vessels, glomus, nerve fibers and the epithelium. Hence, TCs likely serve as transducing centers that provide cell singling via a TPs network to other cell types and structures6.The communication between TCs and stem cells indicated a potential role for TCs in tissue regeneration. A similar conclusion was reported in the heart; after joining with stem cells, TCs are involved in the regeneration and repair of myocardial infarction57. These results may be attributable to hormonal variations between the two seasons. Gonadal activity is increased during the spring and declines during the summer in the camel27. Thus, TCs play an important role in male fertility. The impairment of oviduct TCs leads to infertility58.The present study described for the first-time changes in TCs behavior during the active and inactive breeding seasons in the efferent ductules of the camel. TCs exhibited moderate seasonal differences in their morphological features and activities. Some characteristics (morphology and density) of TCs change with some conditions42 as well as avian species59, reptiles50, amphibians60 and clitellates species2. CD34/PDGFR-α has been identified as a specific marker for TCs61. CD34 is a transmembrane phosphoglycoprotein that identified on hematopoietic stem and other progenitor cells including muscle satellite cells, corneal keratocytes, interstitial cells, epithelial progenitors, and vascular endothelial progenitors62. Vimentin positive TCs are also recognized in various tissues and organs such as uterine tube and uterus63, lung18, placenta64, mammary gland65, and heart66. Vimentin is one of the intermediate filaments provide structural support to maintain cellular integrity and protective function against stress67. Expression of vimentin is associated with normal development, cellular transformation, and growth in tissue culture68. Vimentin is expressed in a wide range of cells; fibroblasts, endothelial cells in blood vessels, epithelial cells, macrophages, cells of cartilage and bone, some vascular smooth muscle cells and melanocytes68. In addition, TCs exhibited stronger immunoreactivity for ESR1, PR and AR in the spring than in the summer. Steroids, particularly estrogen, progesterone and androgen mediated their actions through ESR, PR and AR70. Oestrogen plays a vital role in efferent ductule functions to reabsorb testicular fluid; thus, the lack of ESR1 may lead to a disturbance in the epithelial morphology and inhibit reabsorption of the testicular fluid71. However, progesterone affects male fertility and testosterone biosynthesis72. There is considerable evidence suggesting that, TCs are important targets for steroid hormones. TCs can function as ‘hormonal sensors’ in the human reproductive tract because they express progesterone and estrogen receptors8. Previous studies have reported the presence of ESR and PR on TCs of the myometrium73, fallopian tubes41, Seminal vesicles40. TCs in the fallopian and myometrium tissues may act as steroid sensors74. TCs may be involved in androgens secretion as reported before, they are connected to Leydig cells50.TCs share immunological marker characteristics of undifferentiated stem cells, such as CD34 and vimentin. In the current study, all TCs in the efferent ductules of camel were CD34, VEGF and vimentin positive. Camel TCs express CD34 similar immunophenotype of TCs in other mammalian species75, maintain vascular integrity76 and regulate vascular permeability77. Hence VEGF is termed as the vascular permeability factor (VPF). VEGF increase the vascular permeability by the endothelial cells through increasing the fenestrations78. Both VEGF and PDGF Receptor-α are expressed by TCs80. In the current study, VEGF was also expressed by camel efferent ductule TCs. The seasonal variation of TCs VEGF immunoreactivity was observed. TCs in the inactive summer season express strong immunoreactivity for VEGF more than the active spring season. Diminishing expression of the VEGF in the spring may serve in the thermoregulation that required for spermatogenesis. VEGFA have a significant role in the regulation of vascular permeability as well as in spermiogenesis and the proliferation of spermatogonia82.TCs could play a potential role in the development of blood vessels and endothelium. Vascular endothelial growth factor (VEGF) is one of the signaling protein belongs to the platelet-derived growth factor (PDGF) family. VEGF promote angiogenesis83 demonstrating that VEGF expression in the testis is downregulated during the mating season in one of the seasonal breeder animals, roe deer. Moreover, Camels have adapted to the hypoxic condition of the desert during summer season, in turn the hypoxia activates hypoxia inducible factor 1 (HIF-1). Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that involved in regulation oxygen consumption84. VEGF is one of the downstream gene of the HIF85. This may explain higher expression of the VEGF of TCs in the summer than the spring season.This result agree with earlier report86. TCs acquired signs of exaggerated secretory activities in the spring. TPs became expanded and packed by secretory vesicles. As observed by SEM, a large number of secretory masses were shed from TCs. Some TCs became enlarged in size. It suggests that during the spring season, elevation of the reproductive activity, there was increased demand for extensive transmission of intercellular information that utilizes small molecules, exosomes87. These vesicles may contain various growth factors or cytokines, which are very important in regulating the microenvironmental factors87.Several ultrastructural changes were observed in TCs during the both seasons. TPs in summer had delicate ramifications whereas, TPs in spring may exhibit fine arborization and became more corrugated. TCs, to become able to connect with the other cells or structures through their TPs or by extracellular vesicles and play a potential role in the maintenance of the reproductive tissue homeostasis and renewal88.Intranucleolar chromatin was observed in TCs as well as lymphocytes. The structural organization of intranucleolar chromatin is associated with seasonal and hormonal variation. This finding suggests that the intranucleolar chromatin may act as a temporarily inactive gene that is activated when necessary to satisfy the functional demand in the reproductive cycle particularly the secretory function. Identification of the intranucleolar chromatin composition revealed that they represented as rDNA transcriptional units89.In conclusion, seasonal differences in TCs morphology and behavior are likely regard to hormonal variations between the two seasons. Hormonal alternations during the reproductive cycle may influence the TCs morphology and secretory behavior. The results of the present study support the hypothesis that TCs are affected by microenvironmental changes. TCs gain additional short TPs in an environment characterized by oxidative stress. These cells also change their morphology and acquire a long and slender shape in N-acetyl cysteine cell culture mediumsupplementary figures and legends


Bursaphelenchus xylophilus invasion produce phytoalexins to combat this nematode. Nevertheless, the phytoalexins of Asian pine trees are ineffective against B. xylophilus. The anti-phytoalexin genes of B. xylophilus disable almost all Asian pine phytoalexins, which has allowed B. xylophilus to devastate pine forests in eastern Asia over the last four decades. However, to date, the factors that stimulate anti-phytoalexin gene expression and the mechanisms by which these genes act are not well understood.Pine trees challenged by B. xylophilus using transcriptomic and bioinformatics analyses. The genes that were induced by both Pinus massoniana and carvone and had similarly elevated expression trends were considered anti-phytoalexin genes. Altogether, 187 anti-phytoalexin genes were identified, including 4 cathepsin genes. KEGG pathway enrichment indicated that those cathepsins were related to the Lysosome pathway. Since cathepsins help to maintain metabolic homeostasis by participating in the degradation of heterophagic and autophagic material, the lysosomal cathepsin gene Bx-cathepsin W was cloned and characterized. The results of the RNAi assessment indicated that the knockdown of Bx-cathepsin W reduced the survival rates of B. xylophilus under carvone or P. massoniana stress. The correlation between Bx-cathepsin W and the susceptibility of pines showed that Bx-cathepsin W might help improve the anti-phytotoxin ability of B. xylophilus.Here, we described anti-phytoalexin genes in Bx-cathepsin W supported the survival of B. xylophilus under P. massoniana phytoalexin stress. The cDNA library sequencing, differentially expressed gene identification, and WGCNA algorithm analysis provided insight at a systemic level into the gene regulation of B. xylophilus in response to the immune reaction of P. massoniana. These results will lead to a better understanding of the function of nematode defenses in host innate immunity.The results indicated that the anti-phytoalexin gene  Bursaphelenchus xylophilus, the causative agent of a devastating epidemic of pine wilt disease, is a migratory endoparasite that causes severe damage to pine forests in East Asia and Europe  [dT] 3′) was selected.Fluorescent siRNAs corresponding to Bx-cathepsin W with the primers BxCWqF (TTG CAT TCT ACG GCC AGT CC) and BxCWqR (ACT GAC TTT CGA TGG CTC CG). Bx-Actin RNA was used as an internal control with the primers BxACqF (TTC AGG TGT TAC CCA CAC CG) and BxACqR (GCG GTG GTG GTG AAA GAG TA). Each treatment was performed on three separate biological replicates, and each replicate was measured three times. The quantification and normalization of the data followed the log2fold method according to the Promega instructions. Student’s t-test was used to analyze differences between group grades. The second group was used to assess survival differences. The survival assay of B. xylophilus was performed in 24-well polystyrene culture plates containing 500 μl ddH2O or carvone (800 mg/L) per well in the dark at 25 °C. One thousand nematodes were then added to each well. Moving and immobilized nematodes were counted every 2 days. The third group was used to assess the pathogenicity difference of P. massoniana. RNAi-treated nematodes were inoculated into 3-year-old P. massoniana seedlings  to test the pathogenicity of P. massoniana. The inoculations of M9 buffer-treated nematodes and ddH2O were set as the control. Thirty days after inoculation, nematodes were isolated and counted from P. massoniana seedlings. Three biological replicates were performed for the inoculation.RNAi was performed using mixed stages of nematodes soaked in M9 buffer with 10 mM octopamine and siRNAs (2 μg/μl) or M9 buffer with 10 mM octopamine as a control overnight in the dark at 25 °C, as outlined in Wang, et al. . The RNAB. xylophilus genes. The module to which Bx-cathepsin W belonged and the association between its expression level and the susceptibility of different species of pines were identified. The survival rate of B. xylophilus and the disease susceptibility index of different species of pines  were selected as two traits for WGCNA.Weighted gene coexpression network analysis (WGCNA) is a method frequently used to explore the complex relationships between genes and phenotypes . The disP. massoniana for 1 day and 15 days, P. thunbergii for 1 day and 15 days, P. sylvestris var. mongolica for 15 days, and CK2, a control of nematodes cultured on B. cinerea) were sequenced and analyzed according to the same method described above and were designated as the second sequencing. Two transcriptomes from the first sequencing (Treatment 1 and CK1) and all 6 transcriptomes from the second sequencing were analyzed by weighted gene WGCNA to find clusters (modules) of highly correlated anti-phytoalexin genes .To characterize the anti-phytoalexin gene transcript patterns, 4 cDNA libraries  of An average of 79.69 Mb raw reads was obtained for each sample. After removing the low-quality reads, more than 66.74 Mb clean reads (6.62 Gb clean bases) for each sample were obtained. Clean reads were mapped to the reference genome, the result showed that more than 93% of the transcripts could mapping to the reference genome  but had no homolog in the genome of C. elegans. The 3rd group included 54 genes that had a homolog in the genome of C. elegans. The 4th group included 20 xenobiotic-metabolizing enzyme (XME) genes  database or the genome of Among those 187 anti-phytoalexin genes, 95 genes could be enriched as 66 KEGG orthologs , was cloned from the Treatment 2 cDNA library. Bx-cathepsin W contained a coding sequence of 1179 bp, encoding a polypeptide of 392 amino acids composed of a putative 16-residue signal peptide, 141-residue propeptide, and a 235-residue mature protein. Protein sequence comparisons revealed 77% homology with human cathepsin W  [C. elegans cathepsin F , and approximately 53~75% homology with human cathepsins F, O, X, K, H, and S . The deduced amino acid sequence contained the highly conserved residues of the catalytic triad of papain-like cysteine proteases  , 76% homBx-cathepsin W silencing was found in B. xylophilus after the nematode soaked in siRNAs overnight. The patterns of fluorescein observed for the nematodes indicated that FAM/Cy3 labeled siRNA was taken up by B. xylophilus effectively  and morphology . This result indicated that the resistance of B. xylophilus to carvone decreased after the knockdown of Bx-cathepsin W.However, the survival rate of the Bx-cathepsin RNAi-treated nematodes was found  and pine susceptibility , which indicated that genes in this module were related to the interaction between nematodes and pines. The GS of Bx-cathepsin W with survival rate was 0.79 , the GS of Bx-cathepsin W with pine susceptibility was 0.77 , and the MM of Bx-cathepsin W with yellow module was 0.79 . This result indicated that Bx-cathepsin W had a strong positive correlation with the susceptibility of pines; in other words, Bx-cathepsin W could improve the anti-phytotoxin ability of B. xylophilus, which was consistent with the Bx-cathepsin W RNAi analysis and suggested that Bx-cathepsin W functions as an anti-phytotoxin via an additional method of analysis.The survival rate of ait Fig. a. The moBx-cathepsin W-correlated genes were identified from the yellow module; among them, the connections of the corresponding topological overlap of 439 genes were above a threshold of 0.08  in the pine or the vector Monochamus alternatus. In particular, B. xylophilus must cope with phytoalexins, which are induced by pine in response to nematode infection. Anti-phytoalexin is a crucial process for B. xylophilus to cope with its pine hosts.Furthermore, in order to find a common core among different phytoalexins induced  studies , 32. HowP. massoniana and carvone. However, the detailed functions of these anti-phytoalexin genes require further study. The roles of nematode cathepsins, one proteinase of cysteine proteinases (EC 3.4.22), may include invasion of host tissues, parasite nutrition and evasion of host defense responses [Heterodera glycines [Meloidogyne incognita [Necator americanus [Teladorsagia circumcincta [H. glycines [M. incognita [M. incognita preparasitic infective J2 s is directly related to the parasitic aspects of the plant-nematode interaction [In this study, we identified 187 anti-phytoalexin genes that showed the same promoted expression trends upon induction by both esponses , 34. Catglycines  and Meloncognita . Cysteinericanus  and Telaumcincta . Previouglycines  and M. incognita  females.eraction .Bx-cathepsin W functioned in the anti-phytoalexin process of B. xylophilus in response to P. massoniana and carvone stresses. Carvone-treated B. xylophilus was not strictly correlated with feeding in Treatment 2, and the promotion of Bx-cathepsin W expression indicates that some function of Bx-cathepsin W is indeed more directly related to the plant-nematode interaction, especially to the anti-phytoalexin process. The results of the RNAi assessment indicated that Bx-cathepsin W supported the survival of B. xylophilus under carvone or P. massoniana stress. From the relationship between the expression level of Bx-cathepsin W and the survival rate of B. xylophilus in different pines, we found that the gene expression was positively related to the survival rate of B. xylophilus. Therefore, the correlation of the expression level of Bx-cathepsin W with the susceptibility of pines was examined. These results showed that Bx-cathepsin W played an important role in improving the anti-phytotoxin ability of B. xylophilus. The survival rate of B. xylophilus was positively correlated with the susceptibility of pines; that is, the nematodes that infect more susceptible pines are more likely to survive and therefore are greater in number than nematodes that infect less susceptible pines. Therefore, based on the above results, Bx-cathepsin W is important in the pine wood nematode infection process. According to the correlation between genes in the yellow module, genes that shared the same expression pattern and had a high correlation with Bx-cathepsin W might have a strong regulatory relationship with Bx-cathepsin W, which could indicate a direction for further research on Bx-cathepsin W and a new target for the control of the nematode.Therefore, we were particularly interested in determining whether Bx-cathepsin W, support the survival of the nematode B. xylophilus under P. massoniana phytoalexin stress. The cDNA library sequencing, differentially expressed gene identification and WGCNA algorithm provided insight at a systemic level into the gene regulation of B. xylophilus in response to the immune reaction of P. massoniana. These results will lead to a better understanding of the function of nematode defenses in host innate immunity.The results indicated that the anti-phytoalexin genes, particularly Additional file 1: Table S1. Statistical analysis of the RNA sequencing data. Table S2. Xenobiotic-metabolizing enzyme genes. Table S3. Results of KEGG enrichment. Table S4. Anti-phytoalexin genes related to the Lysosome pathway. Table S5. The number of genes in the 25 modules. Table S6.Bx-cathepsin W-correlated anti-phytoalexin genes. Table S7. Identification of B. xylophilus xenobiotic metabolism genes. Text S1. Transcriptome library preparation and sequencing. Text S2. Data analysis of reads. Text S3. Transcriptome assembly. Text S4. The differential gene expression analysis. Text S5. The WGCNA algorithm.


Telocytes have recently emerged as unique interstitial cells defined by their extremely long, thin and moniliform prolongations termed telopodes. Despite growing evidence that these cells consistently reside in the stromal compartment of various organs from human beings, studies dealing with telocytes in structures of the oral cavity are scarce. Hence, the present morphologic study was undertaken to explore for the first time the presence and specific localization of telocytes within tissues of the normal human tongue, a complex muscular organ whose main functions include taste, speech, and food manipulation in the oral cavity. Telocytes were initially identified by CD34 immunostaining and confirmed by CD34/PDGFRα double immunofluorescence and transmission electron microscopy. CD34+/PDGFRα+ telocytes were organized in interstitial meshworks either in the tongue lamina propria or in the underlying striated muscle. Lingual telocytes were immunonegative for CD31, c-kit and α-SMA. Telopodes were finely distributed throughout the stromal space and concentrated beneath the lingual epithelium and around CD31+ vessels, skeletal muscle bundles/fibers, and intramuscular nerves and ganglia. They also enveloped salivary gland units outside the α-SMA+ myoepithelial cells and delimited lymphoid aggregates. These findings establish telocytes as a previously overlooked interstitial cell population worth investigating further in the setting of human tongue pathophysiology. These cells have been primarily recognized based on their unique ultrastructural morphology as interstitial/stromal cells featuring a small nucleated cell body giving rise to exceptionally long moniliform/varicose cytoplasmic processes (termed ‘telopodes’) with a ‘beads-on-a-string’-like appearance conferred by the alternation of very slender segments and cistern-like dilated portions 10. Afterwards, many efforts have been made to further differentiate TCs from other stromal cell types, mainly fibroblasts, through an in-depth deciphering of their immunophenotypic features and gene expression, proteomic and microRNA profiles15. Though at present TC-specific antigens are still lacking, TCs from virtually every organs (at least in humans) appear to consistently express the cell surface glycoprotein CD34 and, therefore, they are commonly referred to also as TCs/CD34+ stromal cells18. Furthermore, mounting evidence indicates that colocalization of CD34 and platelet-derived growth factor receptor α  is a hallmark of TCs, making them straightforwardly discernible by means of CD34/PDGFRα double immunofluorescence staining22. Telopodes are diffusely distributed and interconnected in the interstitium where they organize three-dimensionally in a labyrinthine meshwork communicating and exchanging signals with a variety of adjacent cell types through both cell-to-cell connections and release of extracellular vesicles29. As exceptional interstitial connecting devices, TCs are supposed to be committed to intercellular signaling likely making a significant contribution to tissue morphogenetic and developmental processes, homeostatic balance, immunosurveillance as well as reparative/regenerative mechanisms by supporting local stem cell niche maintenance and differentiation or performing themselves as progenitors35. Besides tissue physiologic conditions, the notion that TCs may undergo interesting variations in a number of pathologic states suggests either their possible pathogenetic implications or their potentiality as target cells for regenerative medicine purposes42.During the last decade, there has been substantial progress in our understanding of the morphofunctional characteristics of a variety of organs from different systems thanks to the discovery of a previously neglected cell population residing in the stromal space, namely telocytes (TCs)16. As far as the digestive system is concerned, these cells have been uncovered in different portions of the gastrointestinal tract, but, to the best of our knowledge, reports dealing with TCs in structures of the oral cavity are scarce43. Therefore, we undertook this morphologic study to provide a first proof of the existence and microanatomic localization of TCs in tissues of the normal human tongue, a peculiar striated muscular organ covered in oral mucosa with functions as various as taste, speech, food manipulation and swallowing initiation.Thus far, TCs have been broadly described in multiple microanatomic locations, including the connective compartment of various parenchymatous organs, the lamina propria and submucosa of several hollow organs, and the interstitium of all three types of muscle44, we started by studying TCs within tissues of the human tongue by means of immunoperoxidase-based immunohistochemical detection of the CD34 cell surface antigen , exhibited a normal microscopic structure consisting of a mass of interlacing skeletal muscle bundles firmly bound to the overlying oral mucosa by a dense connective tissue , with numerous mucous and serous accessory salivary glands scattered throughout the tongue lamina propria and muscle   Fig. . Immediaes) Fig. . Moreovees) Fig. . CD34 imes) Fig. . As showes) Fig. .Findings of CD34 immunohistochemistry in the tongue muscle are shown in Fig. 17, we first considered that CD34 cannot be assumed as a TC specific marker since it is expressed by at least another type of connective tissue-resident cells, namely CD31+ endothelial cells of blood vessels. On examination of CD34 immunolabeled tissue slides one must therefore pay attention to not misidentify as TCs even the adjacent blood vessels, particularly when the latter are sectioned as thin and elongated vascular profiles with unidentifiable lumen. Hence, CD34/CD31 double immunofluorescence clearly confirmed the presence of a widespread stromal meshwork of CD34+/CD31− TCs around CD34+/CD31+ blood vessels and CD34−/CD31+ lymphatic vessels , c-kit/CD117 and PDGFRα. According to relevant literatureels Fig. . Since iSMA Fig. . Of noteSMA Fig.  or confiSMA Fig. . In addiSMA Fig. . Indeed,SMA Fig. . Keeping1  constitute a previously unrecognized cell meshwork spanning in the whole stromal compartment of the normal human tongue, from the basement membrane underneath the stratified squamous epithelium to the underlying lamina propria and the interstitium of the deeper striated muscle.Although we have recently witnessed the identification and thorough description of TCs/CD34+ stromal cells at different anatomic sites in human beings and other vertebrates44, we found that also in the human tongue the CD34+/PDGFRα+ immunophenotype can be considered a reliable TC hallmark. Our findings also reinforce the growing notion that CD34/CD31 double immunofluorescence should be carried out in this kind of studies as a useful tool to unequivocally differentiate CD34+/CD31+ endothelial cells of blood vessels from the CD34+/CD31− TCs6, that are often concentrated in perivascular locations and may even organize as cell components of the vascular adventitial layer. Moreover, although some authors have pointed out that the thin endothelium of lymphatics might be mistaken with the slender silhouette of TCs51, here we clearly show that this is unlikely by using immunohistochemistry since there is no immunophenotypic overlap between CD34−/CD31+ lymphatic endothelial cells and CD34+/CD31− TCs. On the basis of certain similarities in the ultrastructural morphology of TCs and interstitial cells of Cajal (ICC) as testified by the former name of TCs (i.e. ICC-like cells)3, it has been reported that TCs from some organs may share with the ICC also the expression of c-kit/CD117, though subsequent researches either failed to confirm those findings or demonstrated that TCs are negative for c-kit/CD117 in other organs52. Similarly, here we show that lingual TCs do not express the c-kit/CD117 marker, whose immunoreactivity was detectable only in oval/round-shaped mast cells frequently observed in the close vicinity of the TC cytoplasmic processes. In addition, by means of CD34/α-SMA double immunostaining we could exclude the presence of a ‘myoid’/‘myofibroblast-like’ TC subtype in the tongue stromal compartment. In fact, all TCs within tongue tissues appeared to be CD34+/α-SMA−, and some TCs were concentrated just on the outside of the CD34−/α-SMA+ myoepithelial cell layer surrounding the secretory acini of lingual salivary glands. Of note, these findings are quite similar to those recently reported in the human testis, where CD34+/α-SMA− TCs were seen to form a peritubular outer layer adjacent to an inner layer of CD34−/α-SMA+ ‘myoid’ cells9. On the contrary, the existence of α-SMA+ ‘myoid’ subtypes of TCs has been suggested in the interstitial space of organs of the urinary system, such as the kidney and urinary bladder46. However, it is worth noting that the aforementioned studies46 did not employ CD34/α-SMA double immunofluorescence and, therefore, it cannot be excluded that such α-SMA+ spindle-shaped cells referred to as ‘myoid’ TCs might rather correspond to myofibroblasts, which are well known to express α-SMA53. Even though we frankly acknowledge that a comprehensive ultrastructural analysis of human tongue stromal space was not possible because of the unavailability of specimens of the lamina propria, it is remarkable that we could confirm the presence of cells ultrastructurally identifiable as TCs within the tongue muscle interstitium in the same locations previously disclosed by light microscopy.This study was primarily designed to comprehensively examine TCs by light microscopy through both classic CD34 immunohistochemistry and a number of double immunofluorescence reactions that allowed an in-depth characterization not only of the TC antigenic profiles but also of their specific tissue locations and relationships with neighboring cells. In particular, as exhaustively documented by numerous reports from our and other research groups that have described TCs in different organs56, and such paucity of functional data may be in part ascribed to the fact that protocols for TC isolation are only at early stages, thus hampering reliable in vitro investigations. Nevertheless, we can assume that lingual TCs might play a variety of functions based either on their specific microanatomic locations or on the multiple physiologic roles that have been hypothesized for the TCs in previous works40. As proposed in a number of organs40, it can be speculated that the unique meshwork of TCs extending from the entire lamina propria to the stromal space of the underlying skeletal muscle might have a supportive role during both morphogenesis of the human tongue and post-natal shaping of its extraordinarily complex three-dimensional anatomic structure, that represents a great challenge in the context of regenerative medicine57. The lamina propria fibrous connective tissue provides support and nutrition to the lingual epithelium and, likely, the TC networks bordering the basal epithelial side and surrounding blood and lymphatic vessels may be important mediators of such functions. Even in the underlying tongue muscle interstitium, TCs were distributed to intimately cover both muscle fibers and vascular and nervous structures, making conceivable their involvement in the regulation of muscular trophism and activity. Though we found that lingual TCs are immunonegative for the c-kit/CD117 antigen, we cannot exclude that a subpopulation of these cells might express other stemness markers, possibly representing relatively immature cells/progenitors with regenerative potential59. Of note, the identification of TCs in the tongue connective tissue and striated muscle, that at variance with the mesenchyme-derived tissues in which these cells have previously been analyzed are known to arise embryologically from cranial neural crests61, may raise some interesting questions on the TC embryologic origin, which is worth of future investigations. Taken into account that a recent study has identified subepithelial TCs as a crucial source of Wnt molecular signals without which intestinal crypt stem cells cannot properly proliferate and renew54, it is tempting to speculate that the TC plexus located just beneath the lingual epithelium might perform similarly. Hence, those TCs could be engaged in intercellular signaling supporting the turnover of epithelial cells covering the tongue surface as well as of specialized cells harbored in the taste buds. In such a context, it is worth mentioning that Wnt signaling seems to exert a fundamental role in supporting tissue integrity during tongue development62. Consistent with previous reports on TCs in human exocrine pancreas, parotid gland and labial salivary glands64, we observed that TCs encircle the lingual salivary secretory structures, which might suggest that they are involved in the regulation of glandular functionality in cooperation with the neighboring α-SMA+ myoepithelial cells. Lastly, in line with previous observations in both physiologic and pathologic conditions65, the arrangement of a subset of lingual TCs to precisely delimit and separate subepithelial lymphoid aggregates from the surrounding tissue may be in favor of a participation of those cells in local immunosurveillance.As already discussed, the main goal of the present work was to determine morphologically/immunophenotypically the presence of TCs within the tongue as a hitherto unexplored organ, and therefore we recognize that further analyses will be necessary to definitely shed lights on the precise functions of lingual TCs. Indeed, it should be considered that at present only a few studies have demonstrated with certainty the role of TCs in some organsIn summary, the present findings on the human tongue add to a growing body of data published over the past ten years and strengthen the notion that it is time to reconsider our knowledge of the human microscopic anatomy by recognizing TCs as a distinctive stromal cell identity. However, further studies should be devoted to definitely prove the multiple proposed TC functions and to decipher the mechanisms mediating the cross-talk between TCs and other cell types. Clearly, the ‘strategic’ TC locations that we herein highlighted within the stromal space of the normal human tongue represent the essential groundwork for upcoming functional studies, as well as for future investigations of TCs in different tongue pathologies.67. One paraffin-embedded normal tongue tissue block per case was selected with informed consent to prepare tissue slides for histochemical and immunohistochemical analyses. The study was carried out in accordance with the Declaration of Helsinki and the approval of the institutional review board of Careggi University Hospital, Florence, Italy.Cases were retrieved from the archival files of the Institute of Histopathology and Molecular Diagnosis, Careggi University Hospital, Florence, Italy. A total of 5 consecutive patients  treated for oral cavity squamous cell carcinoma between March and December 2017 were selected. The main criterion for inclusion was the presence of abundant non-pathologic tissue around the tumor. Clinical and pathologic data were retrospectively collected. All patients were treated with complete surgical excision of the tumor with wide margins. Cervical lymph node dissection was also performed in 4 cases based on clinical and imaging evaluation, and all resulted negative. Normal tissue for the study was selected more than 5 mm far from the infiltrative edge of the tumor. This numerical criterion was applied because tissue that is at least 5 mm or more from invasive carcinoma is considered free of tumor cells when the adequacy of surgical margins is evaluated in the pathologic reporti.e. absence of any obvious histopathologic feature).Tongue sections (3 µm thick) from each paraffin-embedded tissue block were deparaffinized and routinely stained with hematoxylin and eosin to confirm the normal tissue appearance  followed by inactivation of endogenous peroxidases. Tissue slides then underwent blockade of non-specific antibody binding sites and overnight incubation at 4 °C with a primary mouse anti-human CD34 antibody. Detailed information on the antibody source and dilution is provided in Table CD34 immunohistochemistry on paraffin-embedded human tissue sections (3 µm thick) was carried out following a standardized protocol employing the ready-to-use UltraVision™ Detection System reagents  that has been described in detail in previous reports from our research group on TC investigation in a variety of human organs68. Briefly, tongue sections were deparaffinized with xylene, rehydrated, unmasked in sodium citrate buffer , and treated with a glycine solution (2 mg/ml) to quench autofluorescence signals before starting the immunostaining procedure. After blockade of non-specific antibody binding sites by applying a solution of 1% bovine serum albumin in PBS for 1 hour at room temperature, tissue slides were incubated overnight at 4 °C with a mixture of mouse and rabbit or goat primary antibodies diluted in blocking solution. Details on primary antibody sources and dilutions are shown in Table http://rsbweb.nih.gov/ij).Paraffin-embedded human tissue sections (3 µm thick) were subjected to double immunofluorescence combining mouse with either rabbit or goat primary antibodies as reported elsewhere10. TCs and telopodes detected in electron microscopy images were digitally colored in blue using Adobe Photoshop CS6 software .Semithin and ultrathin sections  from archival epoxy resin-embedded human tongue muscle specimens were processed according to standardized protocols as detailed elsewhere


Women in nursing professions are at high risk for developing varicose veins as it requires physical work and prolonged standing. The aim of the study is to estimate the current prevalence of varicose veins among nurses at Dhulikhel Hospital and assess its risk factors.A cross sectional study was carried out among 181 female nurses from different clinical settings of Dhulikhel Hospital. A structured questionnaire was administered to gather the demographic, work related and medical history information. The participants underwent Doppler ultrasound for varicose veins confirmation. Varicose veins was defined as Doppler finding of reflux or vein diameter equal or greater than 5 mm.p-value <0.00).A total of 181 nurses participated in this study and 83 (46%) had varicose veins. The mean standing time was 4.28 (0.8) hours /day, mean sitting time was 1.28 (0.6) hours/day, mean walking time was 2.37 (0.8) hours/day. In the adjusted model the odds of having varicose veins was 27 times greater with every 1 hour increase in standing time per day (adjusted OR: 27.44; 95% CI 4.09–180.77; Varicose veins was prevalent among nurses’ at Dhulikhel Hospital. Prolonged standing was found to be a significant factor for varicose veins. Varicose veins are a common chronic venous disorder affecting 20 to 60% of adults worldwide –4. HowevWomen in nursing professions are at high risk for developing varicose veins as their job requires physical work and prolonged standing . AlthougThis cross sectional study was conducted between November 2017 and February 2018 at Dhulikhel Hospital, a Kathmandu University Hospital.We recruited 181 nurses with minimum of 6 months of clinical experience. The participants were all female and included subjects from different clinical work stations of the hospital , and teaching faculties). We excluded anyone if they (a) had surgeries or anesthesia within the past 6 months, (b) neurological conditions, (c) were currently pregnant or pregnant in the past year, and (d) a history of varicose veins prior to starting nursing practice. Prior to the study, we provided the information regarding the study to the participants and obtained their written consent.The demographic information such as age (in years), education (certificate level/ bachelors or above) marital status (married/ not married), weight (in kilograms), height (in meters), Body Mass Index (BMI), parity (nulliparous/ parous).Work related information including clinical experience (in years), clinical work stations, time spent in standing (hours/ day), time spent in sitting (hours/ day) and time spent in walking (hours/ day).Medical history including family history of varicose veins (present/ absent), Bowel movements (Regular/ irregular).We administered a structured questionnaire to measure:The primary outcome was varicose veins. We defined varicose veins based on the Comprehensive classification system for chronic venous disorders (CEAP) . A specip-value. We also conducted the analysis for walking hours with categorical variables (<= 3 h per day/ > 3 h per day) to make it comparable to other studies [Data not shown]. We used SPSS version 21 for the analysis.We summarized the sample characteristics using mean (standard deviation (SD)) for continuous variables and frequency (percentage) for categorical variable. We assessed the association between varicose veins and the nurses’ clinical work stations using chi-square test. We utilized multivariate logistic regression model to assess the association of varicose veins with age, BMI, marital status, parity, education level, bowel habit, family history, work experience, time spent in standing, time spent in sitting and time spent in walking. We presented both crude and adjusted odds ratio (OR) with 95% confidence interval (CI) and 2.Table p = 0.01). The highest prevalence was among teaching faculties who were actively engaged in teaching and the lowest was in subjects in the orthopedic ward. Out-patient departments, surgery ward, and critical care units also had higher prevalence compared to pediatrics ward, medicine ward and orthopedics ward. Figure Nearly three-quarters of the participants had no visible veins present. One-fifth of the participants had a clinical presence of edema at their lower extremity Table . The ovep-value <0.00). In the unadjusted model, age, parity, education and family history were positively associated with varicose veins but the association was not significant after adjusting for socio-demographic variables and working hours’ pattern. Sitting hours and walking hours were negatively associated with varicose veins prevalence. However, these associations were not significant in multivariate model   have been diagnosed with varicose veins when assessed with Doppler ultrasound. Secondly, we were able to control multiple confounding factors in our analysis by using multivariate logistic regression.Our study is not devoid of limitations. Since this is a cross sectional study, both risk factors and varicose veins were measured at one point of time therefore, causality cannot be established. The measurement of hours spent in standing, sitting or walking was self-reported thus the participants might have underestimated or overestimated their standing hours, especially if they had known about their disease condition. This study was conducted among nurses of one center , so the results might not be generalized to other population.Varicose veins were predominantly present with a prevalence of 46% among the nurses at Dhulikhel Hospital. Prolonged standing was found to be a significant risk factor for varicose veins. The nurses should try to avoid unnecessary prolonged standing at work and use sitting or walking whenever possible. This change in clinical practice could ultimately reduce the risk for vascular disease.Additional file 1: Table S1. Factors associated with varicose vein among nurses at Dhulikhel Hospital (n = 181).


Gas-fired electric plants are equipped with heavy machines, which produce hazards including noise pollution. Exposure to high level of noise of above 85dB(A) is known to bring about Noise-Induced Hearing Loss (NIHL). This study aimed to assess noise exposure level and reported prevalence of noise-induced hearing loss among workers in gas-fired electric plants.This cross-sectional study was conducted in three gas-fired electric plants in Dar es Salaam  from July to August 2017. A noise logging dosimeter was used to measure personal noise exposure level. A questionnaire was used to collect information on managerial factors, individual factors, socio-demographic factors and history of the participants. A short screening validated questionnaire was used to obtain noise exposure score. Frequency distribution, Chi-square test and Regression analyses were done using SPSS version 20.One hundred and six participants were involved in the study. Noise exposure level among gas-fired electric plant workers was above 85dB(A), n = 37. The equivalent sound level (LAeq) measured over 8 hours was (98.6 ± 9.7) dB(A). The mean noise peak level was (139.5 ± 9.4) dB(A). Plant C had higher mean noise exposure level (TWA) of (96.9 ± 5.1) dB(A) compared to plant B 96.4 ± 3.7dB(A) and plant A 78.7 ± 11.9dB(A). Participants in both operation and maintenance had higher equivalent sound level (LAeq) measured over eight hours of 101.980 ± 3.6dB(A) compared to maintenance alone 98.5 ± 12.4dB (A) or operation 97.7 ± 8.8dB (A). Proportion of participants with reported hearing loss was 57(53.8%) where 44(41.5%) participants reported difficulty hearing people during conversations. Hearing protective devices (HPDs) were reported to be used by a majority, 101(95.3%).Workers in gas-fired plants are exposed to high noise levels that could damage their hearing. Hearing conservation programs should be established and maintained in this work environment. Noise pollution has been recognized as one of the major threats to human well-being. It has been shown that noise, in extreme ranges, can damage hearing and can be classified as a hazard . Noise, The effect of exposure to noise in relation to the intensity as well as frequency characteristics of the noise was investigated in two Tanzanian textile mills. A peak noise level above the threshold limit value 85dB(A) at a hazardous frequency range of 2500–5000 Hz was recorded . AnotherNoise induced hearing loss (NIHL) generally occurs over a long period of exposure to high noise levels, developing unnoticeably and gradually as time passes . It has A study of electrical workers, often viewed as the least exposed of the trades, resulted in exposures over the OSHA’s Allowable Limit (AL) of 85dB(A) in 25% of dosimeter samples and exceeded the PEL of 90dB(A) in 5% of samples .High prevalence of hearing loss among workers exposed to hazardous noise have been reported in various industries worldwide . It is eAnother cross-sectional study done in the steel industry in Iran showed that 41.3 percent of employees had standard threshold shift in both ears, and there was a significant relationship between the noise exposure level and work experience with standard threshold shift .There is significant association between hearing loss and cardiovascular disease, diabetes, high blood pressure, smoking, lack of exercise and high cholesterol . DiabeteMost occupational diseases are preventable by different methods such as engineering controls, administrative measures and personal protective equipment(PPEs) . However19In Tanzania, noise exposure and the reported prevalence of noise-induced hearing loss among workers in gas-fired electric plants was not known. Therefore, this study aimed to determine employee noise exposure level and reported prevalence of noise-induced hearing loss among workers in gas-fired electric plants so as to put in place proper control strategies. This study may provide information that can be used by other researchers in the future in solving noise exposure problems in the working environment.This was a cross-sectional conducted in three gas-fired electric plants  in Dar es Salaam. Plant A has an installed capacity of 105 Megawatts (MW) consisting of three open cycle gas turbines, SGT–800 industrial type turbines of 35 MW each. Plant B is installed with five generating units giving a total installed capacity of 45 MW. Plant C is installed with twelve generating engines each with a capacity of 8.73 MW totaling an installed capacity of 104 MW. All plants use natural gas in their operations.Participants were randomly selected from administrative list in each plant based on the number of workers in operation and maintenance departments. Proportional sampling method was used to insure weighted participation from each plant. Using OpenEpi software and a proportional of 16% prevalence of noise-induced hearing loss reported by Ivera and colleagues  the totaA questionnaire was used to collect data on socio-demographic factors, managerial factors and individual factors such as use of hearing protection devices, behavior on hearing protection devices, and feeling of hearing loss. A short screening validated questionnaire was used to obtain a noise exposure score . The firNoise exposure scores were divided in to two groups.Noise exposure score in a range of 0–4 is termed as low noise risk, where a participant’s risk of developing noise-induced hearing loss is relatively low if the participant continues to experience similar levels of noise in the future. However, if noise exposures increase, the risk of developing hearing loss will increase as well.Noise exposure score ranging from 5 and above is termed as high noise risk, where a participant is at the risk of developing noise-induced hearing loss if the participant continues to experience similar or higher levels of noise in the future.Noise logging dosimeters were used to measure noise exposure level  among workers in three of the gas-fired electric plants.Data was analyzed using Statistical Package for Social Sciences SPSS version 20. Univariate analysis, bivariate analysis and multiple logistic regression analysis was done. P-value less than 0.05 was considered to indicate statistically significant association between dependent variables and independent variables.A total of 106 workers out of 160 employees in the three gas-fired electric plants participated in this study. Workers in plant A were younger than those in plants B and C. A majority of the participants from the three plants were male (n = 101) (Table The arithmetic mean of noise exposure level (TWA) was 91.3dB(A). The mean equivalent sound level (LAeq) measured over eight hours was (98.6 ± 9.7) dB(A). The mean noise peak level was (139.5 ± 9.4) dB(A). The arithmetic mean score for reported noise exposure score for all the gas-fired electric plant is AM(SD) is 6.9(3.3)  of 101.980 ± 3.6dB(A); the maintenance department had a mean equivalent sound level (LAeq) measured over eight hours of 98.5 ± 12.4dB(A), and the operation department had a mean equivalent sound level (LAeq) measured over eight hours of 97.7 ± 8.8dB(A)  of 98.2 ± 2.3dB(A), whereas the engineers had the lowest noise exposure level (TWA) of 78.5 ± 15.0dB(A). The noise peak level for all the tasks was above 85dBA, which shows that there was no a safe task, even though their noise score and noise exposure level was less than others in comparison (Table Seven (6.6) participants in the gas-fired electric plant were hypertensive. One (0.9) participant had had severe ear infection/injury at childhood. Two (1.9) participants had diabetes. Nine (8.6) participants had experienced ear pain prior to time of the study. One (0.9) participant had used an ototoxic drug, in this case quinine, two weeks before the study Table .Fifty-seven 53.8) reported feeling hearing loss, 44(41) reported to have difficulty in hearing people speaking during conversation, 36(34.0) need to shout in order to be understood when standing three feet away from them, 55(51.9) participants watch people’s faces when they speak to understand what is being said, furthermore 48(45.3) participants reported responding inappropriately in a conversation  among gas-fired electric plant workers. Daily duration of exposure to noise was associated with difficulty in hearing during conversation where p value is 0. 045 (data not shown).Workers in plant C were more likely to report difficulties in hearing conversations compared to workers from plant B and A  adjusted for age, ear pain and habit of attending disco. Furthermore, workers at plant C had higher crude odds ratio  of reporting feeling of hearing loss Table .About 21(19.8) n = 106 participants reported ringing or buzzing sounds in their ears. Higher prevalence of tinnitus was observed among workers in plant C compared to the other plants (p = 0.048) Table .A majority of participants in gas-fired electric plants – 101(95.3) – use hearing protective devices. About 8(7.5) participants use ear plugs, 41(38.7) participants use ear muffs and 57(53.8) participants use both ear plug and ear muff. About 85(80.2) participants had training on the hearing protective devices. Sixty-six (62.3) participants were required by their managers to use hearing protective devices in the gas-fired electric plant. About 84(79.2) participants always use hearing protective devices when working in gas-fired electric plants , 101.8dB(A) and102.6dB(A) in plants A, B and C respectively, above OEL of 85dB(A) set by Tanzanian Bureau of Standards  and the Using a short screening validated noise exposure questionnaire, the mean score for reported noise exposure for all the gas-fired electric plants was AM(SD) was 6.9(3.3). Gas plant C had a higher mean score of 8.59(2.8), whereas gas plant B had a mean noise score of 7.1(2.7) and gas plant A had the mean noise score of 4.5(3.1). A majority of the participants, 74(69.8), had a higher score, showing possible high exposure to noise. Plant A had a few (37.5%) who had a high noise score compared to plants B and C (79%) and (88%), respectively.There was a significant correlation between noise score and noise exposure level among gas-fired electric plant workers (r = 0.409 and p = 0.012). This shows that for developing countries like Tanzania, where there is shortage of personal noise exposure dosimeter, a one-minute validated questionnaire can be used to report noise exposure in occupational settings.Participants in both operation and maintenance departments had a higher equivalent noise exposure level (LAeq) measured over eight hours of 101.980 ± 3.6dB(A) followed by maintenance department had equivalent noise exposure level (LAeq) measured over eight hours of 98.5 ± 12.4dB(A) and operation department had equivalent noise exposure level (LAeq) measured over eight hours of 97.7 ± 8.8dB(A). This has been presented in another study , where mAmong the study participants in gas-fired electric plants, 57(53.8) reported feeling hearing loss. This prevalence is higher than that reported by a study conducted in Dar es Salaam and Morogoro, Tanzania , wherebyAbout 21(19.8) participants reported ringing or buzzing in their ears in the current study, which is low compared to the study done among construction workers in the United States of America, where about 38% indicated they had ringing or buzzing in their ears . This isForty-eight (45.3) participants reported a problem with responding inappropriately during conversation due to a problem of not understanding what people say during conversation in noisy environments. This is low compared to a proportion of 60% who reported a problem understanding what people say in noisy environments in a study done among construction workers in the United States of America . About 4Workers in gas-fired electrical plants are exposed to significant high noise levels, above 85dB(A), which could impair their hearing capacity. Artisans and technicians received significantly higher amounts of exposure than other job categories. The measured noise levels (TWA and peak level) were found to be higher than the TBS and WHO acceptable limit in some production sections. This suggests that specific intervention is required to protect workers exposure to noise and health effects at the work place.


To evaluate the insertion results and hearing preservation of a novel slim modiolar electrode (SME) in patients with residual hearing.(0.125–0.5 kHz) ≤ 80 dB HL). The insertion results were re-examined from the postoperative cone-beam computed tomography scans. Postoperative thresholds were obtained at the time of switch-on of the sound processors (mean 43 days) and at latest follow-up (mean 582 days). The speech recognition in noise was measured with the Finnish matrix sentence test preoperatively and at follow-up.We retrospectively collected the data from the medical files of 17 patients (18 ears) implanted with a SME. All patients had functional low frequency hearing (PTA The mean insertion depth angle (IDA) was 395°. Neither scala dislocations nor tip fold over were detected. There were no total hearing losses. Functional low-frequency hearing was preserved in 15/18 (83%) ears at switch-on and in 14/17 (82%) ears at follow-up. According to HEARRING classification, 55% (10/18) had complete HP at switch-on and 41% (7/17) still at follow-up. Thirteen patients (14 ears) were initially fitted with electric–acoustic stimulation and seven patients (8 ears) continued to use it after follow-up.The preliminary hearing preservation results with the SME were more favorable than reported for other perimodiolar electrodes. The results show that the array may also be feasible for electro-acoustic stimulation; it is beneficial in that it provides adequate cochlear coverage for pure electrical stimulation in the event of postoperative or progressive hearing loss. The preservation of the delicate inner ear structures has become a major consideration in cochlear implant surgery as intracochlear trauma has been shown to negatively affect the post-implant hearing results –5. Due tConventional (i.e. stylet type) perimodiolar electrodes (PME) are reported to cause more trauma as compared to lateral wall electrodes (LWE) , 22, 23.A new PME, the slim modiolar electrode (SME)  was recently designed for atraumatic insertion. The aim of this study was to analyze the clinical insertion and hearing preservation results of the SME.(0.125–0.5 kHz) ≤ 80 dB (HL) were included in this study [We retrospectively collected the data from the medical files of 17 patients (18 ears) implanted with the SME. Patients with relevant functional hearing, defined as preoperative low-frequency PTA is study . Patientis study , 29. RanAll patients underwent routine pre-operative magnetic resonance imaging (MRI) and high-resolution computed tomography (HRCT) to rule out cochlear malformation or retrocochlear pathology. All patients had normal temporal bone and labyrinthine anatomy.The SME is a new generation PME, whose volume is approximately 40% smaller than the Contour array. The internal stylet was replaced by an external sheath to keep the electrode straight prior to its insertion. The SME has a diameter of 0.35 × 0.4 mm at the tip and 0.45 mm × 0.5 mm at the base.All patients underwent cochlear implantation via a trans-mastoid posterior tympanotomy approach under general anesthesia according to the institution’s hearing preservation protocol. The patients were given cefuroxime 1.5 g and dexamethasone 7.5–10 mg intravenously during induction. Weight equivalent doses were administered to the pediatric patients. The bony overhang over the round window (RW) was carefully drilled down to largely expose the round window membrane (RWM). A Spongostan  soaked with dexamethasone 10 mg/ml was placed into the RWM for the time of implant bed drilling. The RWM was incised in the anterior part and lifted posteriorly with a short hook to open the anterior half of the round window. A hyaluronic acid–dexamethasone mixture (50:50 ratio) was then applied onto the RW area. Prior to loading the electrode, the hyaluronic acid–dexamethasone mixture was applied onto the array to ensure smooth gliding of the sheath during insertion. During insertion, special attention was paid to the appropriate orientation of the wing. The insertion and the removal of the sheath were performed as slowly as possible. The final position of the array was finally adjusted, with the distal marker inside the cochlea and the proximal marker outside. The white triangle was locked between the chorda-facial angle and was secured with bone paste and fibrin glue for stabilization. Finally, a tiny piece of temporal fascia was prepared and placed around the array to seal the RW.On the first post-operative day, a cone-beam computed tomography (CBCT) was taken to assess the insertion results. The insertion depth angle (IDA) was measured and the scalar placement was evaluated Fig. . All patThe first postoperative hearing thresholds were mostly measured at the time of switch-on of the sound processor. Thresholds were measured routinely in the follow-up visits at approximately 6 and 12 months after activation.(0.125–0.5 kHz), PTA (0.125–1 kHz) and for HEARRING classification (S) [S = S > 75%, partial S = 75 − 25% and loss when <S 25%. For PTA (0.125-0.5 kHz) and PTA (0.125−1 kHz), complete hearing preservation was achieved when the mean pre- and postoperative threshold deterioration was ≤ 15 dB (HL) and partial hearing preservation when the threshold shift was ≤ 30 dB (HL). A postoperative threshold deterioration > 30 dB (HL) was classified as minimal preservation.The hearing preservation results are presented according to the following classifications used in the literature: The hearing thresholds were analyzed for PTA i et al. . In the Data were analyzed with Statistical Packages for the Social Sciences (SPSS) for Windows version 25 . Wilcoxon signed rank test was used in the statistical analysis when comparing hearing results. The Pearson test was used as a correlation test.All eighteen insertions could be performed through the RWM without any need to drill an extension. All insertions were carried out slowly, over 1 min. There were no post-operative complications. In the post-operative CBCT, the mean IDA was 395 degrees (range 313°–434°). All electrodes were fully inserted without any tip fold-over or scala translocations. The SME was located in close proximity to the modiolus in all but one ear. Information regarding the patient demographics and insertion results is summarized in Table The mean time between surgery and the first postoperative threshold measurements was 43 days . The mean follow-up time for all ears was 582 days .(0.125−0.5 kHz) ≤ 80 dB (HL)) was preserved in 14 out of 17 ears (82%). The mean postoperative deterioration in the PTA (0.125–0.5 kHz) was 11 dB (HL). At end of the follow-up, complete hearing preservation was achieved in 14 out of 17 ears (82.4%) for PTA (0.125–0.5 kHz and in 13 out of 17 ears (76.5%)) for PTA (0.125–1 kHz). Partial preservation was achieved in 1/17 (5.8%) and 3/17 (17.6%) and minimal preservation occurred in 2/17 (11.8%) for PTA (0.125–0.5 kHzand in 1/17 (5.8%)) for PTA (0.125–1 kHz). The corresponding rates for earlier threshold measurements at 3–93 days after surgery, showed a complete preservation rate in 15 out of 18 ears (80.3%) for both PTA (0.125–0.5 kHz) and PTA (0.125–1 kHz). According to the HEARRING classification, 7 out of 17 ears (41%) had complete hearing preservation and 10 out of 17 ears (59%) had partial preservation at the end of follow-up. According to the earlier threshold measurements, complete hearing preservation was present in 55% of the ears and partial preservation in the remaining 45% when applying the HEARRING classification. The hearing preservation results according to the different classifications conducted in the early postoperative period and the final follow-up are summarized in Table There were no total hearing losses. Functional low frequency hearing (PTA 0.15−0.5 kHz0.15−0.5 kHzr = 0.603 (p = 0.01) for PTA (0.125–0.5 kHz) and r = 0.613 (p = 0.009) for PTA (0.125−1 kHz). There was no correlation between the baseline hearing and the preservation after surgery. For PTA (0.125–0.5 kHz), the correlation coefficient was r = − 0.341 (p = 0.180); for PTA (0.125−1 kHz) the value of r was − 0.417 (p = 0.096). We did not detect any significant differences between etiologies and the deterioration of residual hearing at the end of follow-up (PTA125−500 Hzp = 0.768 and for PTA125−1000 Hzp = 0.649).We found a moderate correlation between the patient’s age and the deterioration of the residual hearing at the final follow-up. The correlation coefficients were There were 14 ears (78%) eligible for possible EAS and the vast majority, i.e. 13 ears (72%) were primarily fitted with EAS. Subsequently, eight patients (nine ears) continued to use EAS. Two patients did not experience any subjective benefit from simultaneous acoustic stimulation and three patients preferred an open ear canal to the EAS strategy. The patient with bilateral SMEs used an EAS strategy in both of her ears.p = 0.01) and in the clinically expected magnitude. We found a significant (negative) correlation between IDA and post-operative speech test results , i.e. better post-operative speech test results with deeper IDA. No correlations were detected for the pre- and postoperative SRT values .The mean preoperative SRT was − 1.2 dB (SNR) (range − 6.8 to + 10.0 dB (SNR)). The postoperative SRT improved significantly and was − 5.2 dB (SNR) (range − 8.5 to − 0.7 dB (SNR)). The improvement of Δ − 4.0 dB (SNR) with the Finnish matrix sentence was statistically significant , compared to other LWE’s (approx. 1%) or stylet-type PME’s (approx. 2–3%) , 35–39. In our cohort of patients, we found no scala translocation on the CBCT images and all electrodes were in the scala tympani. McJunkin et al.  describe(0.125–1 kHz) was 12 dB (HL), which is comparable to the value reported by Gantz et al. [(0.125–0.750 kHz) < 15 dB (HL)) results with the SME (50%) and the slim LWE (43%) were similar to our series, whereas very poor hearing preservation (0%) was encountered with the stylet-type PMEs.There are many different classifications for defining postoperative hearing preservation. We chose to present our data according to the most common definitions used in the literature. The hearing preservation rates achieved with the SME appear to be superior to other stylet-type PMEs , 42, 43.z et al. , who fouWhen comparing the results according to the HEARRING classification, complete preservation was observed in 47% of ears. In pediatric patients, Manjaly et al.  reported(0.125−0.5 kHz) and 76.5% for PTA (0.125−1 kHz). Although there is some variation in the methods of assessing low-frequency preservation between different studies, we have achieved comparable short-term preservation results with the SME as reported for LWE. We were not able to measure the thresholds immediately after surgery, which raises the question of whether the threshold shift was due to direct insertion trauma or to postoperative inflammation.LWEs are reported to achieve low frequency hearing preservation in a wide range from 11.3 to 77.7% , 18, 20.We found an age-related effect on the postoperative hearing preservation. Zanetti et al.  reportedWe found a significant improvement in the speech recognition in noise as measured with the FMST. The mean improvement of the speech reception threshold in noise after implantation of 4.0 dB (SRN) was clinically most significant and in the range of the desired and expected improvement . One intThe limitations of this study are inherent in its retrospective nature. The statistical power of the analysis is weakened by the small size and heterogeneity of the cohort. Additionally, our finding should be regarded as preliminary, since a longer follow-up will be needed to evaluate the long-term hearing preservation results.The hearing preservation results with the SME were superior to those reported for stylet type PMEs. In several cases, residual hearing was well preserved which enabled patients to use EAS stimulation. Although the hearing preservation rate of the SME was inferior to that achieved with short LWEs, it provided deeper insertions and better cochlear coverage for pure electrical stimulation in the event of post-operative or progressive hearing loss. This may have obviated the need for re-implantation with a longer electrode in the event of postoperative or progressive hearing loss.


GJB2, SLC26A4, or OTOF. In 6 of 13 families (46%) evaluated using HEar-Seq, deafness was explained by damaging alleles of SLC26A4, MYO15A, OTOG, LOXHD1, and TBC1D24. In some genes critical to hearing, it is particularly difficult to interpret variants that might affect splicing, because the genes are not expressed in accessible tissue. To address this problem for possible splice-altering variants of MYO15A, we evaluated minigenes transfected into HEK293 cells. Results revealed exon skipping in the message of MYO15A c.9083+6T>A, and intron retention in the message of MYO15A c.8340G>A, in each case leading to a premature stop and consistent with co-segregation of homozygosity for each variant with hearing loss. The profile of genetics of hearing loss in this population reflects the genetic heterogeneity of hearing loss and the usefulness of synthetic technologies to evaluate potentially causal variants in genes not expressed in accessible tissues.For multiple generations, much of the Arab population of Northern Israel has lived in communities with consanguineous marriages and large families. These communities have been particularly cooperative and informative for understanding the genetics of recessive traits. We studied the genetics of hearing loss in this population, evaluating 168 families from 46 different villages. All families were screened for founder variants by Sanger sequencing and 13 families were further evaluated by sequencing all known genes for hearing loss using our targeted gene panel HEar-Seq. Deafness in 34 of 168 families (20%) was explained by founder variants in  The identification of alleles causing deafness has advanced dramatically in the past decade as a result of the availability of technologies such as next-generation sequencing (NGS) , 2. ApplSLC26A4, MYO15A, OTOG, LOXHD1, and TBC1D24 that are associated with deafness. In addition, we also examined the consequences of two splicing variants in the MYO15A gene that we identified in these families.Our cohort was comprised of 168 Arab families with non-syndromic hearing loss (NSHL). The members of the population live in 46 different villages in the north of Israel, with long traditions of consanguineous marriage and a long history of endogamy . In thisThis study was approved by the Ethical Committees of the Emek Medical Center and Tel Aviv University, the Helsinki Committee of the Israel Ministry of Health, and by the Human Subjects Division of the University of Washington. A medical history was obtained from all participating individuals, including a detailed pedigree emphasizing hearing loss and other relevant symptoms. Of the 168 families, 151 reported consanguinity. Affected individuals were asked for details about the severity of hearing loss, age of onset, symmetry of hearing loss, use of hearing aids or cochlear implants, experience of tinnitus, exposure to ototoxic drugs and/or noise, pathological conditions of the inner ear, and vestibular function. After acquiring signed informed consent, we obtained fresh blood samples for DNA extraction.GJB2 by Sanger sequencing, using the BigDye Terminator Cycle Sequencing Kit , and analyzed using the ABI PRISM 3100 Genetic Analyzer (Applied Biosystems) according to the manufacturer’s instructions. We then applied massively parallel sequencing (MPS) of deafness genes using the HEar-Seq4 panel, modified from Hear-Seq2 ;[109G>A] and c.[35delG];[298C>T] (2/34). Moreover, we screened for the SLC26A4 c.1197delT variant, as it was previously found in the northern Israel Arab population , 2120, 2In summary, diagnosing the genetic basis of deafness is a first crucial step in understanding the underlying pathological mechanisms causing hearing impairment, and might contribute to our knowledge of the physiological process of hearing. From a clinical perspective, our findings have dramatically improved the clinical management of patients and can be used to guide the genetic counseling provided to affected individuals and their family members. Finally, investigating the consequences of a novel variant may be the cornerstone to developing future gene-specific therapies.Supplementary Table S1Supplementary Table S2


Some immune escape mutants of H9N2 virus and the corresponding mutations in hemagglutinin (HA) have been documented, but little is known about the impact of a single mutation on the antigenicity and pathogenesis of H9N2. In this study, seven critical sites in HA associated with the antigenicity were identified and the effects of a HA mutation (N166D) derived from a H9N2 escape mutant (m3F2) were investigated. Although N166D did not significantly affect viral replication in Madin–Darby canine kidney (MDCK) cells and viral shedding in the larynx and cloaca of chicken, N166D attenuated the pathogenesis of the virus in mice. Compared to the rescued RgPR8-H9_166D, RgPR8-H9_166N caused greater body weight loss and higher viral titers in the lungs of the infected mice. Moreover, hemagglutination inhibition (HI) assay for the sera from the chickens infected with wild type H9N2 and mutant m3F2 showed that N166D mutation could result in weak antibody response in chickens. Considering the field strains of H9N2 with N166D mutation are frequently isolated in the countries with H9N2 vaccination, the findings that the single mutation in HA, N166D, affected both the antigenicity and pathogenesis of H9N2 highlight the significance of surveillance on such mutation that may contribute to the failure of H9N2 vaccination in the field. H9N2 avian influenza virus (AIV) is endemic in Asia, the Middle East, and North Africa, which causes a significant economic loss to the poultry industry due to mild to high morbidity and mortality ,2,3. MorThe identification of novel antigenic sites broadens our knowledge for fully mapping the B cell epitopes in HA of H9N2, and that the single N166D mutation derived from an escape mutant could efficiently result in low antibody response in chickens highlights that such a mutation may contribute to the failure of H9N2 vaccination in the field and the efficient surveillance on such mutation in field strain is critical for better H9N2 vaccine development.2.The H9N2 isolate A/chicken/Jiangsu/XZ299/2016/H9N2 (XZ299) used in this study was propagated in 10-day-old embryonated chicken eggs. Madin–Darby canine kidney (MDCK) cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM), containing 5% fetal calf serum (FCS). Human embryonic kidney 293T cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal calf serum (FCS). These cells were incubated at 37 °C under 5% COH9-specific monoclonal antibodies (MAbs) were generated through the fusion of myeloma Sp2/0 cells with splenocytes from Balb/c mouse immunized with XZ299 virus as previously described . In brieHI assay was performed using 8 hemagglutination units of H9N2 and 0.5% chicken erythrocytes by standard methods as previously described .Selection of escape mutant was described previously . In brie®-LT1 Transfection Reagent . The mixture was incubated at room temperature for 45 min, and then 1 mL of Opti-MEM medium was added. The mixture was then inoculated onto the co-cultured 293T and MDCK cells. After 12 h post-transfection, the medium was changed with 2 mL of fresh Opti-MEM medium with 1μg/mL TPCK-Trypsin. At day 4 post-transfection, the rescued viruses in the supernatant of the transfected cells were collected and titrated in MDCK cells.Viral RNAs of XZ299 and H9N2 mutant were extracted and cDNA was synthesized as previously described . The HA 50 in MDCK cells as previously described [MDCK cells were infected with rgPR8-H9 166N, rgPR8-H9 166D, XZ299, and m3F2 at 0.001 MOI (Multiple of infection), all at 37 °C. The tissue culture supernatants from the infected cells were collected at 12, 24, 36, 48, 60, and 72 h post-infection (hpi), and the viruses were titrated by TCIDescribed .5 TCID50 by intranasal inoculation respectively. At 3 and 6 days post-infection (dpi), three mice from each group were euthanized, and lungs were collected and the virus load in the lungs were titrated by TCID50 in MDCK cells. Six infected mice per group were monitored daily for body weight loss and any clinical signs. The mice with the body weight loss more than 25% will be anesthetized and euthanized.Twelve six-week-old Balb/c mice per group were infected with rgPR8-H9 166N, rgPR8-H9 166D, XZ299, and m3F2 at 105 TCID50 by intranasal inoculation. At 2, 4, 6, 8, and 10 dpi, the swab samples of the larynx and cloaca from six chickens each group were collected and titrated for virus shedding in MDCK cells. At 7 and 14 dpi, six chickens in each group were bled to determine HI titers against XZ299 and H9N2 mutant, respectively.Two-week-old chickens were infected with XZ299 and m3F2 at 10p < 0.05 was considered as a statistically significant difference.Experimental groups were statistically compared using Prism 5.0 software package  to perform an analysis of variance (ANOVA). All animal experiments were performed in accordance with institutional animal care guidelines, and the protocol, #06R015, was approved by the Animal Care Committee at Yangzhou University in China. The ethical permission code  was provided by Animal Ethics Committee of Yangzhou University.Following selection of MAbs, three MAbs  were generated, and ascetic fluids of the MAbs from mice were subsequently produced and harvested. All three MAbs inhibited XZ299 at high HI titers , suggestTo identify novel antigenic sites in HA of H9N2, the HAs of the H9N2 mutants m2G10-1, m2G10-2, m3F2, and m5C7 were sequenced. As shown in To investigate the impact of the N166D mutation on the antigenicity and pathogenesis of H9N2, two viruses, rgPR8-H9 166N and rgPR8-H9 166D, were rescued and the viral growth kinetics were performed. As shown in To evaluate effect of N166D on the viral pathogenesis of H9N2, mice were infected with rgPR8-H9 166D, rgPR8-H9 166N, XZ299, and m3F2 respectively. As shown in To further investigate whether N166D affects antibody response and pathogenesis in chickens, chickens were infected with XZ299 and m3F2. As shown in Mutations in the HA gene of influenza virus can affect both viral antigenicity and pathogenesis, and some HA mutants can efficiently escape from natural or vaccine-induced immunity, which leads to vaccination failure in the field ,8,11. ThAlthough N166D mutation has been identified by others in the mutants with multiple mutations ,14, the In summary, this study is the first demonstration that a single mutation N166D in the escape mutant m3F2 has effects on the antigenicity and pathogenesis of H9N2. Although the N166D mutation attenuates the RP8 backbone-based virus other than the H9N2 backbone-based virus in mice, the N166D mutation does cause a weak antibody response in the chicken. The frequent isolation of the field H9N2 mutants with N166D in the countries with H9N2 vaccination highlights the significance of surveillance on such mutation and development of an efficient H9N2 vaccine.