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AimsGenerally, deciduous and evergreen trees coexist in subtropical forests, and both types of leaves are attacked by numerous insect herbivores. However, trees respond to and defend themselves from herbivores in different ways, and these responses may vary between evergreen and deciduous species. To understand tree responses to leaf herbivore attack under varying intensities of herbivory.MethodsWe examined both the percentage of leaf area removed by herbivores as well as the percentage of leaves attacked by herbivores to evaluate leaf herbivore damage across 15 subtropical deciduous and evergreen trees, quantified plant responses to varying intensities of herbivory and analyzed both leaf herbivore attack and leaf traits among species.ResultsWe have found that there was a higher intensity of herbivory on deciduous species than evergreen species, both as percentage of leaf area removed and the percentage of leaves attacked. In addition, leaf herbivory was positively correlated with defensive traits (concentrations of tannin and lignin), and negatively correlated with nutrient content (ratios of NSC:lignin and NSC:cellulose), which may be largely attributed to long-term and frequent insect herbivore stress and potentially evolutionary arms races between plants and insects.ConclusionsThis study highlights that leaf responses to varying intensities of herbivory differed widely among subtropical plant species and there was a stronger defensive response for deciduous trees to leaf herbivore attack than that of evergreen trees. These results have implications for better understanding plant responses and defenses to varying intensities of herbivory, and reveal several underlying relationships between leaf traits and herbivory.

It has been claimed that salvianolic acid B (Sal B), a natural bioactive antioxidant, exerts protective effects in various types of cells. This study aims to evaluate the antioxidant and anti‐apoptosis effects of Sal B in a cultured HEI‐OC1 cell line and in transgenic zebrafish (Brn3C: EGFP). A CCK‐8 assay, Annexin V Apoptosis Detection Kit, TUNEL and caspase‐3/7 staining, respectively, examined apoptosis and cell viability. The levels of reactive oxygen species (ROS) were evaluated by CellROX and MitoSOX Red staining. JC‐1 staining was employed to detect the mitochondrial membrane potential (ΔΨm). Western blotting was used to assess expressions of Bax and Bcl‐2. The expression pattern of p‐PI3K and p‐Akt was determined by immunofluorescent staining. We found that Sal B protected against neomycin‐ and cisplatin‐induced apoptotic features, enhanced cell viability and accompanied with decreased caspase‐3 activity in the HEI‐OC1 cells. Supplementary experiments determined that Sal B reduced ROS production (increased ΔΨm), promoted Bcl‐2 expression and down‐regulated the expression of Bax, as well as activated PI3K/AKT signalling pathways in neomycin‐ and cisplatin‐injured HEI‐OC1 cells. Moreover, Sal B markedly decreased the TUNEL signal and protected against neomycin‐ and cisplatin‐induced neuromast HC loss in the transgenic zebrafish. These results unravel a novel role for Sal B as an otoprotective agent against ototoxic drug–induced HC apoptosis, offering a potential use in the treatment of hearing loss.

This paper proposes a nonelliptic extended English teaching ability evaluation algorithm based on an adaptive random matrix model. The algorithm models a nonelliptical expansion target as multiple elliptical sub-objectives, and the expansion state of each sub-target is described by an inverse Wishart distribution. The new method is combined to improve the robustness problem caused by the initialization after the algorithm expansion. This research uses the smart teacher education platform to conduct research and analysis on the evaluation of teaching practice ability of intern normal students. From the perspective of data evaluation, we explore the influence of normal students’ curriculum training on the teaching practice ability of normal students. By analyzing the correlation between normal students’ course grades and practice grades, this paper explores the influence of normal students’ prepractice training on normal students’ teaching practice ability. This paper explores the influence of the training of normal students’ course learning on the teaching practice ability of normal students. The results show that the learning level of normal students’ professional courses has a significant impact on the development of normal students’ practice performance and teaching practice ability; the impact of normal students’ pedagogical course level on normal students’ practice performance and teaching practice ability is relatively low.

Nanfeng Zhang,1,* Tingting Qian,2,* Shan Sun,2,* Wei Cao,1 Zhixian Wang,1 Danling Liu,2 Peifan Li,2 Jingfang Wu,2 Huawei Li,1,2 Jianming Yang1 1Department of ENT, Second Affiliated Hospital of Anhui Medical University, Hefei, Anhui, 230031, People’s Republic of China; 2Department of ENT Institute and Otorhinolaryngology, Eye & ENT Hospital, State Key Laboratory of Medical Neurobiology, NHC Key Laboratory of Hearing Medicine Research, Fudan University, Shanghai, 200032, People’s Republic of China*These authors contributed equally to this workCorrespondence: Jianming Yang; Huawei Li, Email JMyang88@163.com; hwli@shmu.edu.cnBackground: Otitis media with effusion (OME) is a non-suppurative inflammation of the middle ear that is characterized by middle ear effusion and hearing loss. However, the mechanisms of OME are not fully understood. The aim of this study was to determine the function and the mechanism of the IL-17 cytokine in the pathogenesis of OME and to investigate IL-17 as a potential strategy for the treatment of OME.Methods: In this study, the OME rat model was induced by ovalbumin (OVA) as previously described. The severity of OME was determined with an oto-endoscope, by histochemical analysis, and by acoustic immittance. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of RNA-sequencing (RNA-seq) data was carried out to analyze the signaling pathways related to the pathogenesis of OME, which indicated that IL-17 is involved in OME. The anti-IL-17A monoclonal antibody was administrated by nasal drip to block IL-17 to treat OME in the rat model. The rats were finally injected intraperitoneally with the inhibitor of Notch signaling pathway to study the mechanisms of IL-17-induced inflammation. Serum and lavage fluid were collected for the detection of related cytokines, and middle ear tissue was collected for Western blot, quantitative real-time PCR (qRT-PCR), and immunohistochemical and immunofluorescence analysis.Results: KEGG analysis of RNA-seq data suggested that the IL-17 signaling pathway might be involved in the onset of OME. IL-17 expression was confirmed to be increased in both the serum and the middle ear of the rat model. The monoclonal antibody against IL-17 neutralized IL-17, inhibited the inflammation in the middle ear, and reduced the overall severity of OME in vivo. Furthermore, the Notch signaling pathway was activated upon IL-17 upregulation in OME and was suppressed by IL-17 blockage. However, there was no change in IL-17 expression after Notch inhibitor treatment, which reduced the severity of OME in the rat middle ear.Conclusion: IL-17 plays a key role in the pathogenesis of the OVA-induced OME rat model. IL-17 induced inflammatory responses via the Notch signaling pathway and targeting IL-17 might be an effective approach for OME therapy.Keywords: otitis media with effusion, IL-17, anti-IL-17A monoclonal antibody, notch

Background: KCNKs, potassium two pore domain channel family K members, can maintain the resting potential, regulate the amplitude and duration of the plateau of the action potential, and change the membrane potential and membrane excitability. Evidence from many studies indicates that KCNKs is abnormally expressed in many solid tumors and plays a regulatory role in the development and malignant progression of cancer. However, the expression pattern and prognostic value of KCNK factors in papillary thyroid carcinoma have not been reported. Methods: In this study, we used the data from databases such as ONCOMINE, GEPIA, Kaplan-Meier Plotter, and cBioPortal to perform bioinformatics analysis of KCNK factors in patients with thyroid cancer. Results: We found that the mRNA expression of KCNK1, KCNK5, KCNK6, KCNK7, and KCNK15 were significantly higher in thyroid cancer tissues than that in normal tissues, while KCNK2, KCNK4, KCNK9, KCNK16 and KCNK17 mRNA levels were decreased compared to normal tissues. And the expression levels of KCNK1/2/4/5/6/7/15 were correlated with the tumor stage. Survival analysis using the Kaplan-Meier Plotter database revealed that KCNK2/3/4/5/12/15 were associated with overall survival (OS) in patients with thyroid cancer. Conclusion: Finally, the results of ROC curves, immunohistochemical staining, immune cell infiltration and kinase / miRNA / transcription factor regulation showed that KCNK2, KCNK4, KCNK5 and KCNK15 levels could be used as biomarkers for PTC diagnosis. This study implied that KCNK2, KCNK4, KCNK5 and KCNK15 are potential targets of precision therapy for patients with thyroid cancer and these genes are new biomarkers for the therapeutic target for thyroid cancer.

Circular RNAs (circRNAs) are novel RNA molecules that play important roles in chemoresistance in different cancers, including breast and gastric cancers. However, whether circRNAs are involved in the response to chemotherapy in small cell lung cancer (SCLC) remains largely unknown. In this study, we observed that cESRP1 (circular RNA epithelial splicing regulatory protein-1) expression was significantly downregulated in the chemoresistant cells compared with the parental chemosensitive cells. cESRP1 enhanced drug sensitivity by repressing miR-93-5p in SCLC. Cytoplasmic cESRP1 could directly bind to miR-93-5p and inhibit the posttranscriptional repression mediated by miR-93-5p, thereby upregulating the expression of the miR-93-5p downstream targets Smad7/p21(CDKN1A) and forming a negative feedback loop to regulate transforming growth factor-β (TGF-β) mediated epithelial-mesenchymal transition. Furthermore, cESRP1 overexpression and TGF-β pathway inhibition both altered tumour responsiveness to chemotherapy in an acquired chemoresistant patient-derived xenograft model. Importantly, cESRP1 expression was downregulated in SCLC patient tissues and was associated with survival. Our findings reveal, for the first time, that cESRP1 plays crucial a role in SCLC chemosensitivity by sponging miR-93-5p to inhibit the TGF-β pathway, suggesting that cESRP1 may serve as a valuable prognostic biomarker and a potential therapeutic target in SCLC patients.

Objectives This study aimed to investigate the clinical manifestations, treatment, and prognosis of traumatic pneumolabyrinth caused by tympanic membrane (TM) perforation. Methods Clinical data were collected from 3 cases of traumatic pneumolabyrinth occurring between 2015 and 2021 and 22 cases were identified from 20 articles in PubMed database that reported pneumolabyrinth due to tympanum-penetrating injury. Intervention Nonoperative treatment was performed in Cases 1 and 3. Middle ear inspection was performed 1 year after the injury due to worsening vertigo upon head movement in Case 2. Main outcome measures Hearing outcomes and vestibular evaluations were presented for the 3 cases, and all comparable cases in the literature were reviewed. Results All 25 patients had a history of traumatic TM perforation, with perforations mostly located in the posterior or posterior superior quadrant (16 cases). Air signs were observed in the vestibule in all 25 patients, 15 of whom revealed stapes luxation into the vestibule. Conservative treatments were performed in 8 cases, and exploratory surgery in 17 cases. Most patients were free of vertigo (23/25). There were no significant hearing improvements in 15 cases, while hearing recovery or improvement was observed in 9 cases. Conclusions The clinical manifestations of pneumolabyrinth due to tympanum-penetrating injuries vary widely. Importantly, the degree of hearing loss is not directly related to the subjectively perceived vertigo but to the location and extent of pneumolabyrinth.

Background: Recent research showed that abnormal lipid metabolism was associated with cancers. As one of the genes that can regulate the level of lipid metabolism, abnormal APOE expression was associated with carcinogenesis. However, the biological role of APOE in papillary thyroid carcinoma (PTC) remains to be determined.Methods: ONCOMINE, GEPIA, UALCAN, STRING, GeneMANIA, LinkedOmics, GSCALite, TISIDB, EPIC and TIMER were utilized to achieve comprehensively bioinformatics analysis of APOE in this study. And the immunohistochemical staining of APOE was used to verify the predicted results.Results: The mRNA level and protein level of APOE of PTC tissues were significantly elevated in TCGA cohort and Shanghai cohort. And APOE expression was positively correlated with the pathological stage and lymph node metastasis in PTC. PTC patients with low mRNA level of APOE were associated with a bad prognosis. The functions of APOE co-expressed genes were mainly enriched in adaptive immune response, protein-lipid complex subunit organization, actin cytoskeleton reorganization, cell chemotaxis, protein activation cascade and transcriptional misregulation in cancer. APOE level was significantly correlated with tumor-infiltrating cells (B cells, CD8+ T cells, neutrophils, and dendritic) and immune biomarkers in PTC.Conclusions: APOE is a potential independent biomarker for PTC and APOE expression is positively correlated with immune cell infiltration in PTC.

Background: In recent years, the annual incidence of thyroid cancer (TC) has increased, with papillary thyroid cancer (PTC) identified as the most commonwinwordpathological type accounting for approximately 80% of all thyroid cancer cases. The tumor microenvironment is known to play a vital role in tumor information transmission and immune detection. Methods: In the present study, we examined gene expression data from 518 patients with PTC. The ESTIMATE algorithm was used to calculate immune and stromal scores of PTC patients. Based on a protein–protein interaction (PPI) network, functional enrichment and overall survival analyses, C-X-C motif chemokine ligand 10 (CXCL10) was identified as a core gene. We further investigated the roles of core genes of PTC in the tumor immune microenvironment using LinkedOmics, GSEA, and TIMER tools. Results: Immune, stromal and ESTIMATE scores were related to clinicopathological variables of patients with PTC, but not survival outcomes. Eight differentially expressed genes (DEGs) were associated with survival outcome. In addition, immunochemical staining experiments revealed lower expression of CXCL10 in PTC than paracancerous tissues. GSEA pathway enrichment analysis revealed downregulation of CXCL10 in multiple cancer pathways. CXCL10 and related genes were enriched in pathways related to adaptive immune response, cellular defense response and regulation of innate immune response. Conclusion: The tumor microenvironment plays a critical role in development of PTC and CXCL10 may serve as a novel target of precision therapy for this patient population.

Sensory hair cells (HCs) are highly susceptible to damage by noise, ototoxic drugs, and aging. Although HCs cannot be spontaneously regenerated in adult mammals, previous studies have shown that signaling pathways are involved in HC regeneration in the damaged mouse cochlea. Here, we used a Notch antagonist (DAPT), a Wnt agonist (QS11), and recombinant Sonic hedgehog (SHH) protein to investigate their concerted actions underlying HC regeneration in the mouse cochlea after neomycin-induced damage both in vivo and in vitro. With DAPT, the numbers of HCs increased, and supporting cell (SC) proliferation was seen in both the intact and damaged cochlear sensory epithelia, while these numbers were unchanged in the presence of QS11. When simultaneously treated with DAPT and QS11, the number of HCs increased dramatically, and much greater SC proliferation was seen in the cochlear epithelium. In transgenic mice with both Notch1 conditional knockout and β-catenin over-expression, cochlear SC proliferation and HC regeneration were more obvious than in either Notch1 knockout or β-catenin over-expressing mice separately. When cochleae were treated with DAPT, QS11, and SHH together, SC proliferation was even greater, and this proliferation was seen in both the HC region and the greater epithelial ridge. High-throughput RNA sequencing was used to identify the differentially expressed genes between all groups, and the results showed that the SHH and Wnt signaling pathways are involved in SC proliferation. Our study suggests that co-regulation of the Notch, Wnt, and SHH signaling pathways promotes extensive cell proliferation and regeneration in the mouse cochlea.

Background:Papillary thyroid carcinoma (PTC) is one of the fastest-growing malignant tumor types of thyroid cancer. Therefore, identifying the interaction of genes in PTC is crucial for elucidating its pathogenesis and finding more specific molecular biomarkers. Methods:In this study, 4 pairs of PTC tissues and adjacent tissues were sequenced using RNA-Seq, and 3745 differentially expressed genes (DEGs) were screened. The results of GO and KEGG enrichment analysis indicate that the vast majority of DEGs may play a positive role in the development of cancer. Then, the significant modules were analysed using Cytoscape software in the protein-protein interaction (PPI) network. Survival analysis, TNM analysis, and immune infiltration analysis of key genes are all analyzed. And the expression of ADORA1, APOE and LPAR5 genes was verified by qPCR in papillary thyroid carcinoma compared to their matching adjacent tissues.Results: A total of 25 genes were identified as hub genes with nodes greater than 10. The expression of 25 key genes in PTC were verified by the GEPIA database, and the overall survival and disease free survival analyses of these key genes were conducted with Kaplan–Meier plots. We found that only three genes were confirmed with our validation and were statistically significant in PTC, namely ADORA1, APOE, and LPAR5. Further analysis found that the mRNA levels and methylation degree of these three genes are significantly correlated with the TNM staging of PTC, and these three genes are related to PTC immune infiltration. Verification of the expression of these three genes by RT-qPCR further confirmed the reliability of our results. Conclusion: Our study identified three genes that may play key regulatory roles in the development, metastasis, and immune infiltration of papillary thyroid carcinoma.Key words :RNA-Seq, papillary thyroid carcinoma, key gene, bioinformatics

Objectives To investigate the role of dibenzazepine (DBZ) in promoting supporting cell (SC) proliferation and hair cell (HC) regeneration in the inner ear. Materials and Methods Postnatal day 1 wild‐type or neomycin‐damaged mouse cochleae were cultured with DBZ. Immunohistochemistry and scanning electron microscopy were used to examine the morphology of cochlear cells, and high‐throughput RNA‐sequencing was used to measure gene expression levels. Results We found that DBZ promoted SC proliferation and HC regeneration in a dose‐dependent manner in both normal and damaged cochleae. In addition, most of the newly regenerated HCs induced by DBZ had visible and relatively mature stereocilia bundle structures. Finally, RNA sequencing detected the differentially expressed genes between DBZ treatment and controls, and interaction networks were constructed for the most highly differentially expressed genes. Conclusions Our study demonstrates that DBZ can significantly promote SC proliferation and increase the number of mitotically regenerated HCs with relatively mature stereocilia bundles in the neonatal mouse cochlea by inhibiting Notch signalling and activating Wnt signalling, suggesting the DBZ might be a new therapeutic target for stimulating HC regeneration., Hair cell (HC) damage is the main cause of permanent hearing loss in mammals. To investigate the role of DBZ in the neonatal mouse cochlea, we cultured postnatal day 1 mouse cochleae with DBZ. We found that DBZ promoted SC proliferation and new HC generation in a dose‐dependent manner in both intact and damaged cochleae. In addition, most of the newly generated HCs induced by DBZ had visible and relatively mature stereocilia bundle structures. Our study demonstrates that DBZ can significantly promote SC proliferation and increase the number of mitotically regenerated HCs with relatively mature stereocilia bundles in the neonatal mouse cochlea by inhibiting Notch signalling and activating Wnt signalling, suggesting the DBZ might be a new therapeutic target for stimulating HC regeneration.

The utricle is one of the five sensory organs in the mammalian vestibular system, and while the utricle has a limited ability to repair itself, this is not sufficient for the recovery of vestibular function after hair cell (HC) loss induced by ototoxic drugs. In order to further explore the possible self-recovery mechanism of the adult mouse vestibular system, we established a reliable utricle epithelium injury model for studying the regeneration of HCs and examined the toxic effects of 3,3′-iminodiproprionitrile (IDPN) on the utriclein vivoin C57BL/6J mice, which is one of the most commonly used strains in inner ear research. This work focused on the epithelial cell loss, vestibular dysfunction, and spontaneous cell regeneration after IDPN administration. HC loss and supporting cell (SC) loss after IDPN treatment was dose-dependent and resulted in dysfunction of the vestibular system, as indicated by the swim test and the rotating vestibular ocular reflex (VOR) test. EdU-positive SCs were observed only in severely injured utricles wherein above 47% SCs were dead. No EdU-positive HCs were observed in either control or injured utricles. RT-qPCR showed transient upregulation ofHes5andHey1and fluctuating upregulation ofAxin2andβ-cateninafter IDPN administration. We conclude that a single intraperitoneal injection of IDPN is a practical way to establish an injured utricle model in adult C57BL/6J micein vivo. We observed activation of Notch and Wnt signaling during the limited spontaneous HC regeneration after vestibular sensory epithelium damage, and such signaling might act as the promoting factors for tissue self-repair in the inner ear.

Prostate cancer-associated ncRNA transcript 6 (PCAT6) is a long intergenic noncoding RNA that is involved in the progression of prostate and lung cancer, acting as a potential diagnostic and prognostic biomarker in nonsmall cell lung cancer. However, little is known about PCAT6 expression and its clinical significance in colon cancer. Here, we aimed to investigate the clinical significance of PCAT6 in colon cancer and its underlying mechanism. The expression of PCAT6 was analyzed in colon cancer tissues using public databases, and a series of in vitro and in vivo experiments was performed to investigate the biological functions of PCAT6 in colon cancer cells and the underlying mechanisms. Our results demonstrated that PCAT6 was upregulated in colon cancer tissues compared with that in noncancerous tissues, correlating with poorer clinical stages and a worse survival status. In vitro and in vivo experiments illustrated PCAT6 promoted cell growth and inhibited cell apoptosis in colon cancer. Mechanistically, PCAT6 enhanced the coenrichment of EZH2 and H3K4me3 at the apoptosis repressor with caspase recruitment domain (ARC) genomic region, promoting the transcriptional activity of ARC. Our data highlighted that PCAT6 acts as a key activator of ARC expression by forming a complex with EZH2, inhibiting cell apoptosis and contributing to colon cancer progression. These findings elucidated that PCAT6 may be a novel prognostic predictor and therapeutic target of colon cancer.

Trefoil factor 3 (TFF3) was found to be overexpressed in many types of tumours. Evidence has shown that TFF3 plays an important role in tumour proliferation, migration and invasion metastasis. However, the impact of TFF3 on patients' clinicopathological characteristics and underlying mechanisms remain unknown in papillary thyroid carcinoma (PTC). In this study, the expression of TFF3 and the epithelial-mesenchymal transition (EMT) transcriptional factor Snail in PTC and para-carcinoma specimens was evaluated by immunohistochemistry (IHC) and Western blot, and the possible associations with lymph node (LN) metastasis and other clinicopathological parameters were analysed. In vitro, the effect of TFF3 on the malignant behaviour of TPC-1 cells was evaluated by cell proliferation assays, cell adhesion assays, colony formation assays, wound-healing assays and transwell chamber invasion assays. EMT markers and regulatory molecules were detected by quantitative RT-PCR (qRT-PCR) and Western blot analysis in the TFF3-knockdown groups and shRNA control group. The results showed that TFF3 was upregulated in PTC tissue and was associated with lymph node metastasis (P=0.0001), pathological grade (P=0.0002) and Snail expression (P=0.0001). The knockdown of TFF3 markedly inhibited the abilities of TPC-1 cell proliferation, adhesion, colony formation, migration and invasion. Mechanically, the results demonstrated that TFF3 might activate the MAPK/ERK signalling pathways, affect the expression of the transcription factors snail and slug in addition to affecting EMT associated markers E-cadherin and N-cadherin, and accelerate the progression of EMT in TPC-1 cells. These findings indicate that TFF3 might promote the metastatic potential of PTC by promoting the EMT process through cascades of MAPK/ERK pathways.

The generation of hair cells (HCs) from the differentiation of proliferating supporting cells (SCs) appears to be an ideal approach for replacing lost HCs in the cochlea and is promising for restoring hearing after damage to the organ of Corti. We show here that extensive proliferation of SCs followed by mitotic HC generation is achieved through a genetic reprogramming process involving the activation of β-catenin to upregulate Wnt signaling, the deletion of Notch1 to downregulate Notch signaling, and the overexpression of Atoh1 in Sox2(+) SCs in neonatal mouse cochleae. We used RNA sequencing to compare the transcripts of the cochleae from control mice and from mice with β-catenin activation, Notch1 deletion, and β-catenin activation combined with Notch1 deletion in Sox2(+) SCs. We identified the genes involved in the proliferation and transdifferentiation process that are either controlled by individual signaling pathways or by the combination of Wnt and Notch signaling. Moreover, the proliferation of SCs induced by Notch1 deletion disappears after deleting β-catenin in Notch1 knock-out Sox2(+) cells, which further demonstrates that Notch signaling is an upstream and negative regulator of Wnt signaling. SIGNIFICANCE STATEMENT We show here that the extensive proliferation of supporting cells (SCs) and the subsequent mitotic hair cell (HC) generation is achieved through a genetic reprogramming process involving activation of β-catenin to upregulate Wnt signaling, deletion of Notch1 to downregulate Notch signaling, and overexpression of Atoh1 in Sox2(+) SCs in neonatal mice cochleae. By comparing the transcripts of the cochleae among controls, β-catenin activation, Notch1 deletion, and β-catenin activation combined with Notch1 deletion group, we identified multiple genes involved in the proliferation and transdifferentiation process that are either controlled by individual signaling pathways or by the combination of Wnt and Notch signaling. This provides a better understanding of the mechanisms behind mitotic HC generation and might provide new approaches to stimulating mitotic HC regeneration.

Supplementary Materials and Methods. (DOCX 19 kb)

Yanqin Sun,1,* Bingshuang Hu,2,* Yanhong Wang,3 Zhen Li,1 Jingfang Wu,4 Yunchu Yang,4 Yue Wei,5 Xiaofeng Peng,5 Hongling Chen,5 Rongqi Chen,5 Pingyan Jiang,5 Sixian Fang,5 Zhiwu Yu,6 Linlang Guo4 1Department of Pathology, Guangdong Medical University, Dongguan, China; 2Department of Radiotherapy, Zhongshan People’s Hospital, Zhongshan, China; 3Department of Gynecology, Jinxiang People’s Hospital, Jining, China; 4Department of Pathology, Zhujiang Hospital, Southern Medical University, Guangzhou, China; 5College of Pharmacy, Guangdong Medical University, Dongguan, China; 6Division of Laboratory Science, Affiliated Cancer Hospital and Institute of Guangzhou Medical University, Guangzhou, China *These authors contributed equally to this work Objective: microRNAs are regulatory molecules regarded as important in the pathogenesis of different types of tumors. microRNA-216a (miR-216a-5p) has been identified as a tumor suppressor in multiple malignancies. However, the role of miR-216a-5p in the pathogenesis of small cell lung cancer (SCLC) remains obscure. The objective of this study was to investigate the role of the miR-216a-5p/Bcl-2 axis in SCLC pathogenesis. Materials and methods: All the experimental methods used were as follows: microarray analysis, cell culture, transient, and stable gene transfection; real-time fluorescence PCR; Western blot; flow cytometry for cell cycle analysis; in vitro proliferation assay; in vitro wound healing experiment; in vivo xenograft model in nude mice; and dual luciferase reporter assay. All statistical analyses were carried out using GraphPad Prism 7 software. Statistical significance was analyzed by Student’s t-test or one-way ANOVA. P

Objectives The posterior lateral line (PLL) system in zebrafish has recently become a model for investigating tissue morphogenesis. PLL primordium periodically deposits neuromasts as it migrates along the horizontal myoseptum from head to tail of the embryonic fish, and this migration requires activity of various molecular mechanisms. Histone deacetylases (HDACs) have been implicated in numerous biological processes of development, by regulating gene transcription, but their roles in regulating PLL during embryonic development have up to now remained unexplored. Material and methods In this study, we used HDAC inhibitors to investigate the role of HDACs in early development of the zebrafish PLL sensory system. We further investigated development of the PLL by cell-specific immunostaining and in situ hybridization. Results Our analysis showed that HDACs were involved in zebrafish PLL development as pharmacological inhibition of HDACs resulted in its defective formation. We observed that migration of PLL primordium was altered and accompanied by disrupted development of PLL neuromasts in HDAC inhibitor-treated embryos. In these, positions of PLL neuromasts were affected. In particular, the first PLL neuromast was displaced posteriorly in a treatment dose-dependent manner. Primordium cell proliferation was reduced upon HDAC inhibition. Finally, we showed that inhibition of HDAC function reduced numbers of hair cells in PLL neuromasts of HDAC inhibitor-treated embryos. Conclusion Here, we have revealed a novel role for HDACs in orchestrating PLL morphogenesis. Our results suggest that HDAC activity is necessary for control of cell proliferation and migration of PLL primordium and hair cell differentiation during early stages of PLL development in zebrafish.

Cisplatin is widely used for chemotherapy of a variety of malignancies. However, the clinical application of cisplatin is hampered by the resultant irreversible hearing loss due to hair cell apoptosis. To date, no practical regimen to resolve this has been developed. Meanwhile, the role of microRNA in protecting hair cells from cisplatin-induced apoptosis in the inner ear has not been extensively investigated. In this study, we monitored miR-183, -96, and -182 turnover in the cochlea during cisplatin treatment in vitro. We found that overexpression of miR-182, but not miR-183 and -96, improved hair cell survival after 3 μM cisplatin treatment in vitro. We demonstrated that overexpression of miR-182 repressed the intrinsic apoptotic pathway by inhibiting the translation of FOXO3a. Our study offers a new therapeutic target for alleviating cisplatin-induced hair cell apoptosis in a rapid and tissue-specific manner.

This work sought to determine the crosstalk between the Notch and Wnt signaling pathways in regulating supporting cell (SC) proliferation and hair cell (HC) regeneration in mouse utricles. We cultured postnatal day (P)3 and P60 mouse utricles, damaged the HCs with gentamicin, and treated the utricles with the γ-secretase inhibitor DAPT to inhibit the Notch pathway and with the Wnt agonist QS11 to active the Wnt pathway. We also used Sox2-CreER, Notch1-flox (exon 1), and Catnb-flox (exon 3) transgenic mice to knock out the Notch pathway and activate the Wnt pathway in Sox2+ SCs. Notch inhibition alone increased SC proliferation and HC number in both undamaged and damaged utricles. Wnt activation alone promoted SC proliferation, but the HC number was not significantly increased. Here we demonstrated the cumulative effects of Notch inhibition and Wnt activation in regulating SC proliferation and HC regeneration. Simultaneously inhibiting Notch and overexpressing Wnt led to significantly greater SC proliferation and greater numbers of HCs than manipulating either pathway alone. Similar results were observed in the transgenic mice. This study suggests that the combination of Notch inhibition and Wnt activation can significantly promote SC proliferation and increase the number of regenerated HCs in mouse utricle.

To investigate serum vascular endothelial growth factor-C (VEGF-C), VEGF-D and VEGFR-3 levels in patients with papillary thyroid carcinoma (PTC) and analyze their relation with the clinicopathological and thyroid function of the patients.Serum samples and the data of thyroid function were collected from 55 patients with PTC and 24 with benign thyroid tumor (BT). ELISA was used to detect VEGF-C/D and VEGFR-3 concentration in the serum samples and their relation with the thyroid function was analyzed.The VEGF-C and VEGFR-3 levels were significantly higher in PTC group than in BT group (P0.05), but VEGF-D level was comparable between them (P0.05). In PTC patients, the elevation of serum VEGF-C and VEGFR-3 levels was associated with an advanced clinical stage (III-IV), elevated thyroid-stimulating hormone (TSH) level, an age over 45 years, and a tumor diameter exceeding 2 cm (P0.05 or P0.01). Patients with lymph node metastasis had significantly higher VEGF-C level but lower VEGF-3 level than those without metastasis regardless of gender. Serum VEGF-D level was higher in PTC patients with lymph node metastasis (P0.05) and elevated TSH level (P0.01) without association with the clinical stage, tumor diameter, age, or gender. The area under ROC curve (AUC) of serum VEGF-C, VEGFR-3 and TSH was 0.803, 0.734 and 0.707 respectively (P0.01), and that of VEGF-D was 0.556 (P0.05); when combined, serum VEGF-C, VEGFR-3 and TSH showed an AUC of 0.862 (P0.01).Detecting serum VEGF-C and VEGFR-3 levels combined with TSH may enhance the early diagnosis rate of papillary thyroid carcinoma.

The activation of cochlear progenitor cells is a promising approach for hair cell (HC) regeneration and hearing recovery. The mechanisms underlying the initiation of proliferation of postnatal cochlear progenitor cells and their transdifferentiation to HCs remain to be determined. We show that Notch inhibition initiates proliferation of supporting cells (SCs) and mitotic regeneration of HCs in neonatal mouse cochlea in vivo and in vitro. Through lineage tracing, we identify that a majority of the proliferating SCs and mitotic-generated HCs induced by Notch inhibition are derived from the Wnt-responsive leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5(+)) progenitor cells. We demonstrate that Notch inhibition removes the brakes on the canonical Wnt signaling and promotes Lgr5(+) progenitor cells to mitotically generate new HCs. Our study reveals a new function of Notch signaling in limiting proliferation and regeneration potential of postnatal cochlear progenitor cells, and provides a new route to regenerate HCs from progenitor cells by interrupting the interaction between the Notch and Wnt pathways.

To investigate the levels of galectin-3 in the serum and surgical specimens from patients with malignant and benign thyroid lesions and explore their clinical significance.Serum samples were collected from 159 patients with thyroid neoplasms and 16 normal subjects for detection of galectin-3 level using ELISA. The expressions of galectin-3 protein and mRNA were also detected in the surgical specimens by immunohistochemistry and in situ hybridization.Galectin-3 protein and mRNA were expressed at a rate of 96.67% in 66 cases of papillary thyroid carcinoma, 83.33% in 6 cases of follicular thyroid carcinoma, 16.67% in 36 cases of papillary hyperplasia, and 11.74% in 51 cases of thyroid adenoma. The expression level of galectin-3 mRNA was slightly lower than but positively correlated with its protein expression. Patients with thyroid carcinoma had significantly higher serum concentration of galectin-3 than those with benign thyroid lesions (papillary hyperplasia and thyroid adenoma) and normal subjects (P0.001). In patients with papillary thyroid carcinoma, galectin-3 positivity in the tumor tissue was associated with a significantly higher serum galectin-3 level in comparison with the negative cases (P0.05).Detection of galectin-3 in both the serum and surgical specimens can improve the diagnosis rate of thyroid carcinoma.

Doxorubicin (Dox) could trigger a large amount of apoptotic cells in the myocardium, which leads to dilated cardiomyopathy and heart failure. S-propargyl-cysteine (SPRC), a producing agent of endogenous hydrogen sulfide (H2S), possesses cardioprotective efficacy. However, the specific effect and mechanism of SPRC in Dox-induced cardiotoxicity remain elusive. Given gp130 with its main downstream signaling molecule, signal transducer and activator of transcription 3 (STAT3), is involved in cardiac myocyte survival and growth; the present study was performed to elucidate whether SPRC counteracts Dox-induced cardiotoxicity, and if so, whether the gp130/STAT3 pathway is involved in this cardioprotective activity. SPRC stimulated the activation of STAT3 via gp130-mediated transduction tunnel in vitro and in vivo. In Dox-stimulated cardiotoxicity, SPRC enhanced cell viability, restored expression of gp130/STAT3-regulated downstream genes, inhibited apoptosis and oxidative stress, and antagonized mitochondrial dysfunction and intracellular Ca2+ overload. Intriguingly, blockade of gp130/STAT3 signaling abrogated all these beneficial capacities of SPRC. Our findings present the first piece of evidence for the therapeutic properties of SPRC in alleviating Dox cardiotoxicity, which could be attributed to the activation of gp130-mediated STAT3 signaling. This will offer a novel molecular basis and therapeutic strategy of H2S donor for the treatment of heart failure.

The stability and hydrolysis kinetics of a phosphate prodrug, adefovir dipivoxil, in solid formulations were studied. The stability relationship between five solid formulations was explored. An autocatalytic mechanism for hydrolysis could be proposed according to the kinetic behavior which fits the Prout-Tompkins model well. For the classical kinetic models could hardly describe and predict the hydrolysis kinetics of adefovir dipivoxil in solid formulations accurately when the temperature is high, a feedforward multilayer perceptron (MLP) neural network was constructed to model the hydrolysis kinetics. The build-in approaches in Weka, such as lazy classifiers and rule-based learners (IBk, KStar, DecisionTable and M5Rules), were used to verify the performance of MLP. The predictability of the models was evaluated by 10-fold cross-validation and an external test set. It reveals that MLP should be of general applicability proposing an alternative efficient way to model and predict autocatalytic hydrolysis kinetics for phosphate prodrugs.

Inhibition of protein neddylation, particularly cullin neddylation, has emerged as a promising anticancer strategy, as evidenced by the antitumor activity in preclinical studies of the Nedd8-activating enzyme (NAE) inhibitor MLN4924. This small molecule can block the protein neddylation pathway and is now in clinical trials. We and others have previously shown that the antitumor activity of MLN4924 is mediated by its ability to induce apoptosis, autophagy and senescence in a cell context-dependent manner. However, whether MLN4924 has any effect on tumor angiogenesis remains unexplored. Here we report that MLN4924 inhibits angiogenesis in various in vitro and in vivo models, leading to the suppression of tumor growth and metastasis in highly malignant pancreatic cancer, indicating that blockage of angiogenesis is yet another mechanism contributing to its antitumor activity. At the molecular level, MLN4924 inhibits Cullin–RING E3 ligases (CRLs) by cullin deneddylation, causing accumulation of RhoA at an early stage to impair angiogenic activity of vascular endothelial cells and subsequently DNA damage response, cell cycle arrest and apoptosis due to accumulation of other tumor-suppressive substrates of CRLs. Furthermore, we showed that inactivation of CRLs, via small interfering RNA (siRNA) silencing of its essential subunit ROC1/RBX1, recapitulates the antiangiogenic effect of MLN4924. Taken together, our study demonstrates a previously unrecognized role of neddylation in the regulation of tumor angiogenesis using both pharmaceutical and genetic approaches, and provides proof of concept evidence for future development of neddylation inhibitors (such as MLN4924) as a novel class of antiangiogenic agents.

The recent genome-wide association study identified a link between vitiligo and genetic variants in the ribonuclease T2 (RNASET2) gene; however, the functional roles of RNASET2 in vitiligo pathogenesis or in melanocyte apoptosis have yet to be determined. The current study was designed to investigate the vitiligo-related expression pattern of RNASET2 and its molecular function involving apoptosis-related signaling proteins and pathways. The results showed overexpression of RNASET2 in epidermis specimens from 40 vitiligo patients compared with that from matched healthy controls. In addition, in vitro analyses indicated that overexpression of RNASET2 was inducible in cultured primary human melanocytes and keratinocytes by stress conditions, that is, exposure to UV irradiation, hydrogen peroxide, and inflammatory factors, respectively, and led to increased cell apoptosis via the tumor necrosis factor receptor-associated factor 2 (TRAF2)–caspases pathway through the physical interaction of RNASET2 with TRAF2. Thus, RNASET2 may contribute to vitiligo pathogenesis by inhibiting TRAF2 expression and, as such, RNASET2 may represent a potential therapeutic target of vitiligo.

An earring compatible telephone handset receiver (20) having a side sound outlet (22) and a center sound outlet (24) located on the receiver's front surface. A selection switch (38) is used to determine either the side sound outlet (22), the center sound outlet (24), or both of the side and center sound outlets (22 and 24) to be active for transmitting sound. When the side sound outlet (22) is active and aligned with the user's ear canal (202), the receiver (20) is kept away from the user's ear lobe (206) and the attached earring (204) to accommodate uses wearing earrings. When the center sound outlet (24) is active, the receiver (20) can be used in its normal way to accommodate users without earrings.