DCLK1 mediated cooperative acceleration of EMT by avian leukosis virus subgroup J and Marek's disease virus via the Wnt/ß-catenin pathway promotes tumor metastasis.

Co-infection with oncogenic retrovirus and herpesvirus significantly facilitates tumor metastasis in human and animals. Co-infection with avian leukosis virus subgroup J (ALV-J) and Marek's disease virus (MDV), which are typical oncogenic retrovirus and herpesvirus, respectively, leads to enhanced oncogenicity and accelerated tumor formation, resulting in increased mortality of affected chickens. Previously, we found that ALV-J and MDV cooperatively promoted tumor metastasis. However, the molecular mechanism remains elusive. Here, we found that doublecortin-like kinase 1 (DCLK1) mediated cooperative acceleration of epithelial-mesenchymal transition (EMT) by ALV-J and MDV promoted tumor metastasis. Mechanistically, DCLK1 induced EMT via activating Wnt/ß-catenin pathway by interacting with ß-catenin, thereby cooperatively promoting tumor metastasis. Initially, we screened and found that DCLK1 was a potential mediator for the cooperative activation of EMT by ALV-J and MDV, and enhanced cell proliferation, migration, and invasion. Subsequently, we revealed that DCLK1 physically interacted with ß-catenin to promote the formation of the ß-catenin-TCF4 complex, inducing transcription of the Wnt target gene, c-Myc, promoting EMT by increasing the expression of N-cadherin, Vimentin, and Snail, and decreasing the expression of E-cadherin. Taken together, we discovered that jointly activated DCLK1 by ALV-J and MDV accelerated cell proliferation, migration and invasion, and ultimately activated EMT, paving the way for tumor metastasis. This study elucidated the molecular mechanism underlying cooperative metastasis induced by co-infection with retrovirus and herpesvirus. IMPORTANCE: Tumor metastasis, a complex phenomenon in which tumor cells spread to new organs, is one of the greatest challenges in cancer research and is the leading cause of cancer-induced death. Numerous studies have shown that oncoviruses and their encoded proteins significantly affect metastasis, especially the EMT process. ALV-J and MDV are classic tumorigenic retrovirus and herpesvirus, respectively. We found that ALV-J and MDV synergistically promoted EMT. Further, we identified the tumor stem cell marker DCLK1 in ALV-J and MDV co-infected cells. DCLK1 directly interacted with ß-catenin, promoting the formation of the ß-catenin-TCF4 complex. This interaction activated the Wnt/ß-catenin pathway, thereby inducing EMT and paving the way for synergistic tumor metastasis. Exploring the molecular mechanisms by which ALV-J and MDV cooperate during EMT will contribute to our understanding of tumor progression and metastasis. This study provides new insights into the cooperative induced tumor metastasis by retroviruses and herpesviruses.

[Retracted] p68 prompts the epithelial­mesenchymal transition in cervical cancer cells by transcriptionally activating the TGF­ß1 signaling pathway.

[This retracts the article DOI: 10.3892/ol.2017.7552.].

Nup210 promotes colorectal cancer progression by regulating nuclear plasma transport.

The nuclear pore complex (NPC) regulates nucleoplasmic transport, transcription and genomic integrity in eukaryotic cells. However, little is known about how NPC works in cancer. In this study, we investigated the role of the nuclear pore protein 210 (Nucleoporin 210, Nup210) in colorectal cancer (CRC). Bioinformatics analysis revealed that the expression of Nup210 was increased in CRC and was associated with poor patient prognosis, but it was not a statistically significant independent prognostic factor. Moreover, knockdown of Nup210 in CRC cells inhibited the proliferation, invasion and metastasis of CRC cells in vivo and in vitro. Additionally, nuclear size and nuclear plasma material transport capacity decreased along with the number and density of NPCs on the surface of CRC cells when Nup210 expression was inhibited. Furthermore, Nup210 required nuclear localization sequences (NLS) to localize to the nuclear membrane surface and interact with importin α/ß, which in turn affected the transit of nuclear plasma material. Importazole, a small molecule inhibitor of importin, along with therapy that targets the Nup210 protein is anticipated to be a novel strategy for CRC treatment. Their combination may be able to more effectively lower CRC tumor load. In conclusion, Nup210 modulates cellular nucleoplasmic transport capability and cell surface NPC density via NLS, thus promoting CRC progression. This discovery validates the molecular function of NPC in the development of CRC and provides a theoretical foundation for NPC-regulated nuclear import targeting as a therapeutic strategy for CRC.

m6A RNA methyltransferase METTL16 induces Cr(VI) carcinogenesis and lung cancer development through glutamine biosynthesis and GLUL expression.

Hexavalent chromium [Cr(VI)] exposure increases the risk of cancer occurrence. This study found that the levels of an atypical methyltransferase, METTL16 were greatly upregulated in the cells, and mouse tissues with Cr(VI) exposure, and played a critical role in cell proliferation and tumor growth induced by Cr(VI). Similarly, we found METTL16 was upregulated in various human cancer tissues. To understand mechanism of METTL16 in inducing carcinogenesis and cancer development, we identified that glutamate-ammonia ligase (GLUL) as the METTL16 functional target for regulating glutamine metabolism and tumorigenesis induced by Cr(VI) exposure. We demonstrated that METTL16 promoted GLUL expression in a m6A-dependent manner. Furthermore, METTL16 methylated the specific stem-loop structure of GLUL transcript, thereby increased the recognition and splicing of pre-GLUL RNA modified site by m6A reader YTHDC1, which ultimately accelerated the production of mature GLUL mRNA. Animal model of Cr(VI) exposure further confirmed that the expression levels of METTL16 and GLUL were both significantly induced in vivo, and there had a significant positive correlation between METTL16 and GLUL levels. Furthermore, we found that YTHDC1 was also important in inducing GLUL expression, and MYC was the upstream mediator of METTL16 to increase its transcriptional activation. Our study revealed new mechanism of metal carcinogenesis and cancer development.

Discovery of a Potent, selective and orally bioavailable CDK9 degrader for targeting transcription regulation in Triple-Negative breast cancer.

Triple-negative breast cancer (TNBC) is a highly aggressive, heterogeneous and invasive subtype of breast cancer with very limited effective modalities of treatment. Degrading the critical transcription regulator cyclin-dependent kinase 9 (CDK9) by proteolysis targeting chimeras (PROTACs) has shown promising potential for treating TNBC. However, to date, CDK9-targeting PROTACs for oral administration in treatment of cancers have not been reported. We herein present the design, synthesis, and extensive biological evaluation of a series of novel PROTACs as orally bioavailable, potent and selective degraders of CDK9 for targeting transcription regulation in triple-negative breast cancer. The developed compound 29 exhibited a desired potency (DC50 = 3.94 nM) with high efficacy (Dmax = 96 %) on CDK9 degradation, and effectively inhibited the proliferation of TNBC MDA-MB-231 cells. Mechanistic investigations revealed that compound 29 is a bona fide CDK9 degrader and can substantially downregulate the downstream targets c-Myc and MCL-1. Furthermore, compound 29 displayed favorable oral bioavailability in mice, and oral administration of degrader 29 significantly depleted CDK9 protein in TNBC tumor tissues and exhibited tumor growth inhibition in TNBC xenograft mice models. Collectively, our work established that degrader 29 is a highly potent and selective degraders of CDK9 with satisfactory oral bioavailability, which holds promising potential for the treatment of TNBC.

Achieving functional recovery without anatomical repositioning: A case of corneal flap inversion trauma post femtosecond laser-assisted in situ keratomileusis surgery.

Post femtosecond laser-assisted in situ keratomileusis corneal flap inversion trauma is an exceptionally rare and challenging complication in ophthalmology, highlighted by a case involving a 29-year-old woman who suffered an eye injury from ice chips 10 months after undergoing bilateral femtosecond laser-assisted in situ keratomileusis surgery. The injury led to a corneal flap inversion and melting, exacerbated by a delay in seeking treatment due to the pandemic. When she finally presented for medical attention, her vision was severely compromised, with dense epithelial ingrowth obstructing the visual axis or blocking the pupillary entrance. Despite these complications, medical interventions to remove the epithelial cells and attempts to reposition the corneal flap were carried out. Although the flap could not be fully anatomically restored due to scarring and stromal melt, the patient experienced a remarkable recovery in vision, achieving 20/17 visual acuity 6 months post-surgery. This case illustrates that satisfactory visual outcomes can be achieved even when perfect anatomical restoration is not possible. It challenges the traditional focus on anatomical perfection in corneal surgery and highlights the potential for functional recovery to take precedence over anatomical correction, reducing the need for further risky procedures. The outcome stresses the importance of tailored patient management and careful assessment of the risks and benefits in complex surgical cases.

Medical imaging for the diagnosis, recurrence and metastasis evaluation of clear cell sarcoma.

Clear cell sarcoma (CCS) of soft tissue is extremely rare, accounting for approximately 1% of all soft tissue tumours. It is very difficult to diagnose CCS based on clinical manifestations. Magnetic resonance imaging (MRI) provides high-resolution images of soft tissues and pathological features such as mucus, necrosis, bleeding, and fat through high and low signals on T1 weighted image (T1WI) and T2 weighted image (T2WI). On the other hand, the paramagnetism of melanin in CCS shortens the relaxation time of T1 and T2, and high signal intensity on T1WI and low signal intensity on T2WI can be found. This is different from most other soft tissue sarcomas. At present, the treatment method for CCS is surgical resection. MRI can effectively display the tumour edge, extent of surrounding oedema, and extent of fat involvement, which is highly important for guiding surgical resection and predicting postoperative recurrence. As an invasive sarcoma, CCS has a high risk of metastasis. Regardless of the pathological condition of the resected tumour, MRI or computed tomography (CT) should be performed every 1-2 years to assess recurrence at the primary site and to screen for metastasis in the lungs, liver, and bones. If necessary, PET-CT can be performed to evaluate the overall condition of the patient.

RNA methyltransferase NSUN2-mediated m5C methylation promotes Cr(VI)-induced malignant transformation and lung cancer by accelerating metabolism reprogramming.

Hexavalent chromium [Cr(VI)], one common environmental contaminant, has long been recognized as a carcinogen associated with lung cancer, but roles and mechanisms of Cr(VI)-induced epigenetic dysregulations in carcinogenesis remain to be investigated. In this study, we identified that RNA m5C methyltransferase NSUN2 was significantly upregulated in Cr(VI)-transformed cells and lung tissues of Cr(VI)-exposed mice. Inhibition of NSUN2 reduced cell proliferation, migration, colony formation and tube formation abilities. We found NSUN2-mediated m5C modification induced metabolic reprogramming and cell cycle by promoting the mRNA stabilities of ME1, GLUT3 and CDK2. In addition, knockdown of NSUN2 attenuated tumorigenesis and angiogenesis in vivo. RNA m5C reader ALYREF was identified to be involved in NSUN2-mediated m5C modification in Cr (VI)-induced carcinogenesis. Further study showed that EP300 induced NSUN2 upregulation through transcriptional activation by inducing histone modification at H3K27ac site for regulating Cr(VI) carcinogenesis. Our findings demonstrated novel role and mechanism of NSUN2 and epigenetic changes by increasing the RNA m5C modification that are important for Cr (VI)-induced carcinogenesis through NSUN2/ALYREF pathway. NSUN2, ALYREF, ME1, GLUT3 or/and CDK2 may be used as potential new biomarkers or/and therapeutic target(s) in the future.

Can unstimulated whole salivary flow objectively classify salivary gland secretory function in Sjögren's syndrome?

INTRODUCTION: The aim of this study is to investigate whether the testing time for unstimulated whole salivary flow (UWSF) can be shortened to 5 min in patients with suspected Sjögren's syndrome (SjS); and which SjS patients can use UWSF to evaluate salivary gland (SG) secretory function. METHOD: A diagnostic cohort comprising suspected SjS patients was conducted to investigate the correlation between UWSF measurements taken at 10 min (UWSF_10 min) and those taken at 5 min (UWSF_5 min). A group of SjS patients was enrolled for a comparison between UWSF and stimulated whole salivary flow (SWSF). RESULTS: In 734 suspected SjS patients, there was a remarkably high concordance between UWSF_10 min and UWSF_5 min (ICC 0.970, P < 0.001; r 0.973, P < 0.001). Reducing the testing time for UWSF to 5 min resulted in a high PPV of 83.8% and an exceptionally high NPV of 98.7%. In 408 SjS patients, the cut-off values of UWSF_10 min were investigated to classify SG secretory function. Using a threshold of > 0.2 mL/min (36.8%, 150/408) instead of SWSF > 0.7 mL/min (indicating mild secretory hypofunction), the specificity and PPV were found to be 94.2% and 94.0%, respectively; and using a threshold of < 0.05 mL/min (16.9%, 69/408) instead of SWSF ≤ 0.7 mL/min (indicating moderate to severe secretory hypofunction), the specificity was remarkably high at 97.6%, accompanied by a high PPV of 91.3%. CONCLUSIONS: This study supports the possibility of reducing UWSF testing time to 5 min; and the SWSF test may be skipped for SjS patients with USWF > 0.2 mL/min, indicating mild secretory hypofunction, or < 0.05 mL/min, indicating moderate to severe secretory hypofunction. Key Points •A diagnostic cohort of 734 patients with clinical suspicion of SjS provides compelling evidence for the potential to reduce the testing time for UWSF from 10 to 5 min. •Our finding challenges the 2019 treatment recommendation for SjS, which does not require SWSF measurement in SjS patients with UWSF ≥ 0.1 mL/min. •We propose that it may be feasible to consider utilizing UWSF instead of SWSF test for objective classification of SG secretory function in over half of SjS patients.

[Application of high-throughput drug sensitivity testing in children with relapsed and refractory acute leukemia]. / é«˜é€šé‡è¯æ•æ£€æµ‹æŠ€æœ¯åœ¨å„¿ç«¥å¤å‘éš¾æ²»æ€§æ€¥æ€§ç™½è¡€ç— ä¸­çš„åº”ç”¨.

OBJECTIVES: To explore the current application of high-throughput drug sensitivity (HDS) testing in children with relapsed and refractory acute leukemia (RR-AL) and analyze the feasibility of salvage treatment plans. METHODS: A retrospective collection of clinical data from children with RR-AL who underwent HDS testing at the Department of Children's Hematology and Oncology of the First Affiliated Hospital of Zhengzhou University from November 2021 to October 2023 was conducted, followed by an analysis of drug sensitivity results and treatment outcomes. RESULTS: A total of 17 children with RR-AL underwent HDS testing, including 7 cases of relapsed refractory acute myeloid leukemia and 10 cases of relapsed refractory acute lymphoblastic leukemia. The detection rate of highly sensitive chemotherapy drugs/regimens was 53% (9/17), while the detection rate of moderately sensitive chemotherapy drugs/regimens was 100% (17/17). Among the 17 RR-AL patients with highly and moderately sensitive chemotherapy drugs and regimens, the MOACD regimen (mitoxantrone + vincristine + cytarabine + cyclophosphamide + dexamethasone) accounted for 100%, with the highest inhibition rate for single-agent mitoxantrone (94%, 16/17), and the highest inhibition rate for targeted therapy being bortezomib (94%, 16/17). Nine patients adjusted their chemotherapy based on HDS testing results, with 4 undergoing hematopoietic stem cell transplantation. Four patients achieved disease-free survival, while 5 died. Eight patients received empirical chemotherapy, with 2 undergoing hematopoietic stem cell transplantation; 4 achieved disease-free survival, while 4 died. CONCLUSIONS: HDS testing can identify highly sensitive drugs/regimens for children with RR-AL, improving the rate of re-remission and creating conditions for subsequent hematopoietic stem cell transplantation.

Roflumilast Attenuates Microglial Senescence and Retinal Inflammatory Neurodegeneration Post Retinal Ischemia Reperfusion Injury Through Inhibiting NLRP3 Inflammasome.

Purpose: Retinal ischemia-reperfusion (RIR) injury is implicated in various retinal diseases, leading to retinal ganglion cells (RGCs) degeneration. Microglial senescence exacerbates inflammation, contributing to neurodegeneration. This study aimed to investigate the potential therapeutic role of Roflumilast (Roflu) in ameliorating microglial senescence and neuroinflammation following RIR injury. Methods: C57BL/6J mice underwent RIR surgery, and Roflu treatment was administered intraperitoneally. BV2 microglial cells were subjected to oxygen-glucose deprivation and reoxygenation (OGD/R) to simulate ischemic conditions in vitro. SA-ß-gal staining was used to detect cellular senescence. Quantitative PCR and ELISA were used to examine the levels of senescence-associated secretory phenotype (SASP) factors. Hematoxylin and eosin (H&E) staining was performed on retinal sections to assess retinal morphology and thickness. Surviving RGCs were labeled and quantified in retinal whole-mounts using immunofluorescence (IF). Furthermore, Western blot and IF staining were used to quantify the proteins associated with the cell cycle and NLRP3 inflammasomes. Results: Roflu treatment reduced microglial senescence, ROS production, and secretion of pro-inflammatory cytokines in OGD/R-exposed BV2 cells. It also restored cell proliferation capacity and reversed OGD/R-induced cell cycle arrest. In vivo, Roflu alleviated retinal senescence, preserved retinal thickness, and protected against RGCs death in the RIR mouse model. Mechanistically, Roflu inhibited the NLRP3 inflammasome activation and suppressed DNA damage signaling pathway in microglia. Conclusions: Roflu exerts neuroprotective effects by mitigating microglial senescence and inflammation via inhibition of the NLRP3 inflammasome in RIR injury. These findings suggest that Roflu may serve as a promising therapeutic strategy for retinal diseases associated with ischemic injury by targeting microglial senescence.

Targeting Lactate: An Emerging Strategy for Macrophage Regulation in Chronic Inflammation and Cancer.

Lactate accumulation and macrophage infiltration are pivotal features of both chronic inflammation and cancer. Lactate, once regarded merely as an aftereffect of glucose metabolism, is now gaining recognition for its burgeoning spectrum of biological roles and immunomodulatory significance. Recent studies have evidenced that macrophages display divergent immunophenotypes in different diseases, which play a pivotal role in disease management by modulating macrophage polarization within the disease microenvironment. The specific polarization patterns of macrophages in a high-lactate environment and their contribution to the progression of chronic inflammation and cancer remain contentious. This review presents current evidence on the crosstalk of lactate and macrophage in chronic inflammation and cancer. Additionally, we provide an in-depth exploration of the pivotal yet enigmatic mechanisms through which lactate orchestrates disease pathogenesis, thereby offering novel perspectives to the development of targeted therapeutic interventions for chronic inflammation and cancer.

Astrocytic P2X7 receptor regulates depressive-like behavioral reactions of mice in response to acute stressful stimulation.

Acute stress causes depressive-like reactions in the tail suspension (TST) and forced swim tests (FST) of mice. Similarly, inescapable foot shock is able to promote the development of anhedonia as indicated by decreased sucrose consumption of treated mice in the sucrose preference test (SPT). The astrocyte-specific deletion of the P2X7R by a conditional knockout strategy or its knockdown by the intracerebroventricular (i.c.v.) delivery of an adeno-associated virus (AAV) expressing P2X7R-specific shRNA in astrocytes significantly prolonged the immobility time in TST and FST. In contrast, the shRNA-induced downregulation of the P2X7R in neurons, oligodendrocytes, or microglia had no detectable effect on the behavior of treated mice in these tests. Moreover, sucrose consumption in the SPT was not altered following inescapable foot shock treatment in any of these cell type-specific approaches. Immunohistochemistry indicated that the administered astrocyte-specific AAV efficiently conveyed expression of shRNA by hippocampal CA1 astrocytes, but not by neurons. In conclusion, P2X7R in astrocytes of this area of the brain appears to be involved in depressive-like reactions to acute stressors.

Innovative In Situ Passivation Strategy for High-Efficiency Sb2(S,Se)3 Solar Cells.

An effective defect passivation strategy is crucial for enhancing the performance of antimony selenosulfide (Sb2(S,Se)3) solar cells, as it significantly influences charge transport and extraction efficiency. Herein, a convenient and novel in situ passivation (ISP) technique is successfully introduced to enhance the performance of Sb2(S,Se)3 solar cells, achieving a champion efficiency of 10.81%, which is among the highest recorded for Sb2(S,Se)3 solar cells to date. The first principles calculations and the experimental data reveal that incorporating sodium selenosulfate in the ISP strategy effectively functions as an in situ selenization, effectively passivating deep-level cation antisite SbSe defect within the Sb2(S,Se)3 films and significantly suppressing non-radiative recombination in the devices. Space-charge-limited current (SCLC), photoluminescence (PL), and transient absorption spectroscopy (TAS) measurements verify the high quality of the passivated films, showing fewer traps and defects. Moreover, the ISP strategy improved the overall quality of the Sb2(S,Se)3 films, and fine-tuned the energy levels, thereby facilitating enhanced carrier transport. This study thus provides a straightforward and effective method for passivating deep-level defects in Sb2(S,Se)3 solar cells.

Intercellular adhesion molecule-1 (ICAM-1): From molecular functions to clinical applications in cancer investigation.

Intercellular adhesion molecule-1 (ICAM-1) is a versatile molecule that plays a critical role in various physiological and pathological processes, particularly in tumor development where its impact is bidirectional. On the one hand, it augments the immune response by promoting immune cell migration, infiltration, and the formation of immunological synapses, thus facilitating potent antitumor effects. Simultaneously, it contributes to tumor immune evasion and influences metastasis by mediating transendothelial migration (TEM), epithelial-to-mesenchymal transition (EMT), and epigenetic modification of tumor cells. Despite its significant potential, the full clinical utility of ICAM-1 has yet to be fully realized. In this review, we thoroughly examine recent advancements in understanding the role of ICAM-1 in tumor development, its relevance in predicting therapeutic efficacy and prognosis, as well as the progress in clinical translational research on anti-ICAM-1-based therapies, encompassing including monoclonal antibodies, immunotherapy, antibody-drug conjugate (ADC), and conventional treatments. By shedding light on these innovative strategies, we aim to underscore ICAM-1's significance as a valuable and multifaceted target for cancer treatment, igniting enthusiasm for further research and facilitating translation into clinical applications.

Induced pluripotent stem cell-derived mesenchymal stem cells enhance acellular nerve allografts to promote peripheral nerve regeneration by facilitating angiogenesis.

Previous research has demonstrated the feasibility of repairing nerve defects through acellular allogeneic nerve grafting with bone marrow mesenchymal stem cells. However, adult tissue-derived mesenchymal stem cells encounter various obstacles, including limited tissue sources, invasive acquisition methods, cellular heterogeneity, purification challenges, cellular senescence, and diminished pluripotency and proliferation over successive passages. In this study, we used induced pluripotent stem cell-derived mesenchymal stem cells, known for their self-renewal capacity, multilineage differentiation potential, and immunomodulatory characteristics. We used induced pluripotent stem cell-derived mesenchymal stem cells in conjunction with acellular nerve allografts to address a 10 mm-long defect in a rat model of sciatic nerve injury. Our findings reveal that induced pluripotent stem cell-derived mesenchymal stem cells exhibit survival for up to 17 days in a rat model of peripheral nerve injury with acellular nerve allograft transplantation. Furthermore, the combination of acellular nerve allograft and induced pluripotent stem cell-derived mesenchymal stem cells significantly accelerates the regeneration of injured axons and improves behavioral function recovery in rats. Additionally, our in vivo and in vitro experiments indicate that induced pluripotent stem cell-derived mesenchymal stem cells play a pivotal role in promoting neovascularization. Collectively, our results suggest the potential of acellular nerve allografts with induced pluripotent stem cell-derived mesenchymal stem cells to augment nerve regeneration in rats, offering promising therapeutic strategies for clinical translation.

Imaging findings of isolated congenital middle ear malformation on high-resolution computed tomography.

PURPOSE: This study aims to analyze the imaging features of isolated congenital middle ear malformation (CMEM) on high-resolution computed tomography (HRCT). METHODS: We retrospectively collected patients with surgically confirmed diagnosis of isolated CMEM in our hospital between January 2018 and June 2023. All patients underwent HRCT before surgery. The preoperative imaging findings were analyzed by neuroradiologists with full knowledge of the intraoperative findings. RESULTS: 37 patients were included in this study, including 25 males and 12 females, with a median age of 16 years. A total of 44 ears underwent surgery. The most commonly affected structures were incudostapedial joint, incus long process, and stapes superstructure, followed by stapes footplate, oval window, incudomalleolar join, tympanic segment of the facial nerve canal, incus body, incus short process and malleus. All incus defect/hypoplasia/malposition, stapes superstructure deformity, malleus deformity, incudostapedial joint discontinuity, and facial nerve canal malposition/abnormal bifurcation could be observed on HRCT. Additionally, 96.0% of stapes superstructure defect, 85.7% of oval window atresia, and 41.7% of incudomalleolar joint fusion, could be visualized on HRCT. HRCT could not show ossicular soft tissue pseudo-connection and stapes footplate fixation. CONCLUSIONS: Preoperative HRCT is an important tool for diagnosing isolated CMEM. The advantages of HRCT lie in its ability to detect ossicular defects/deformities, incudostapedial joint discontinuity, oval window atresia, and facial nerve abnormalities. However, it has a low detection rate for incudomalleolar joint fusion and cannot show ossicular soft tissue pseudo-connection and stapes footplate fixation.

Genome-wide analysis of the AP2/ERF gene family in Pennisetum glaucum and the negative role of PgRAV_01 in drought tolerance.

APETALA2/ethylene-responsive (AP2/ERF) plays crucial roles in resisting diverse stresses and in regulating plant growth and development. However, little is known regarding the structure and function of the AP2/ERF genes in pearl millet (Pennisetum glaucum). The AP2/ERF gene family may be involved in the development and maintenance of P. glaucum resilience to abiotic stresses, central to its role as a vital forage and cereal crop. In this study, PgAP2/ERF family members were identified and comprehensive bioinformatics analyses were performed, including determination of phylogenetic relationships, gene structures, conserved motifs, chromosomal localization, gene duplication, expression pattern, protein interaction network, and functional characterization of PgRAV_01 (Related to ABI3/VP1). In total, 78 PgAP2/ERF members were identified in the P. glaucum genome and classified into five subfamilies: AP2, ERF, DREB, RAV, and soloist. Members within the same clade of the PgAP2/ERF family showed similar gene structures and motif compositions. Six duplication events were identified in the PgAP2/ERF family; calculation of Ka/Ks values showed that purification selection dominated the evolution of PgAP2/ERFs. Subsequently, a potential interaction network of PgAP2/ERFs was generated to predict the interaction relationships. Additionally, abiotic stress expression analysis showed that most PgAP2/ERFs were induced in response to drought and heat stresses. Furthermore, overexpression of PgRAV_01 negatively regulated drought tolerance in Nicotiana benthamiana by reducing its antioxidant capacity and osmotic adjustment. Taken together, these results provide valuable insights into the characteristics and functions of PgAP2/ERF genes, with implications for abiotic stress tolerance, and will ultimately contribute to the genetic improvement of cereal crop breeding.