OBJECTIVES: The purinergic receptor P2X2 (P2RX2) encodes an ATP-gated ion channel permeable to Na⁺, K⁺, and especially Ca²⁺. Loss-of-function mutations in P2RX2 are known to cause autosomal dominant nonsyndromic deafness 41 (DFNA41), which manifests as high-frequency hearing loss, accelerated presbycusis, and increased susceptibility to noise-induced damage. Zebrafish, owing to their small size, rapid development, high fecundity, transparent embryos, and high gene conservation with humans, provide an ideal model for studying human diseases and developmental mechanisms. This study aims to generate a p2rx2 knockout zebrafish model using CRISPR/Cas9 gene editing system to investigate the effect of p2rx2 deficiency on the auditory system, providing a basis for understanding P2RX2-related hearing loss and developing gene therapy strategies. METHODS: Two CRISPR targets (sgRNA1 and sgRNA2) spaced 47 bp apart were designed within the zebrafish p2rx2 gene. Synthesized sgRNAs and Cas9 protein were microinjected into single-cell stage Tübingen (TU)-strain zebrafish embryos. PCR and gel electrophoresis verified editing efficiency at 36 hours post-fertilization (hpf). Surviving embryos were raised to adulthood (F0), tail-clipped, genotyped, and screened for positive mosaics. F1 heterozygotes were generated by outcrossing, and F2 homozygous mutants were obtained by intercrossing. Polymerase chain reaction (PCR) combined with sequencing verified mutation type and heritability. At 5 days post-fertilization (dpf), YO-PRO-1 staining was used to examine hair cell morphology and count in lateral line neuromasts and the otolith region. Auditory evoked potential (AEP) thresholds at 600, 800, 1 000, and 2 000 Hz were measured in nine 4-month-old wild type and mutant zebrafish per group. RESULTS: A stable p2rx2 knockout zebrafish line was successfully established. Sequencing revealed a 66 bp insertion at the first target site introducing a premature stop codon (TAA), leading to early termination of protein translation and loss of function. Embryos developed normally with no gross malformations. At 5 dpf, mutants exhibited significantly reduced hair cell density in the otolith region compared with wild type, although lateral line neuromasts were unaffected. AEP testing showed significantly elevated auditory thresholds at all 4 frequencies in homozygous mutants compared with wild type (all P<0.001), indicating reduced hearing sensitivity. CONCLUSIONS We successfully generated a p2rx2 loss-of-function zebrafish model using CRISPR/Cas9 technology. p2rx2 deficiency caused hair cell defects in the otolith region and increased auditory thresholds across frequencies, indicating its key role in maintaining zebrafish auditory hair cell function and hearing perception. The phenotype's restriction to the otolith region suggests tissue-specific roles of p2rx2 in sensory organs. This model provides a valuable tool for elucidating the molecular mechanisms of P2RX2-related hearing loss and for screening otoprotective drugs and developing gene therapies.
Animals ; Zebrafish/genetics* ; Receptors, Purinergic P2X2/deficiency* ; CRISPR-Cas Systems/genetics* ; Gene Knockout Techniques ; Phenotype ; Zebrafish Proteins/genetics* ; Disease Models, Animal

To investigate the clinical features, early recognition and prognosis of influenza-associated intracranial aspergillosis. The clinical data of a child with influenza-related intracranial aspergillosis admitted to the Department of Pediatrics of the Third Xiangya Hospital of Central South University in 2022 were retrospectively analyzed. By searching CNKI, Wanfang Database, Web of Science, PubMed and other Chinese and English databases as of November 2023, literature was screened according to inclusion and exclusion criteria, and the clinical characteristics, diagnosis, treatment and prognosis of all influenza-related intracranial aspergillus disease were summarized. A total of 5 reports of 5 cases of influenza-associated aspergillosis were obtained, including 1 child patient and 1 child influenza-associated intracranial aspergillosis in this study, and a total of 6 influenza-associated aspergillosis patients. The clinical manifestations of the 6 patients were disturbance of consciousness, hemiplegia, pupil changes, behavioral abnormalities, epilepsy, etc. Most of the cases were diagnosed through pathology or autopsy to find pathogens. In this case, the pathogens were confirmed through metagenomic next-generation sequencing (mNGS). Antifungal drugs were the first choice for treatment. 66.7% of the surviving patients received surgical treatment, and the mortality rate of 6 patients reached 50.0%. Influenza-associated intracranial aspergillosis is rare, can occur in both children and adults, and has a poor prognosis. In the early stage of influenza, if there are neurological symptoms, screening for fungal infection should be conducted. Acer second generation sequencing can improve the diagnosis rate of intracranial aspergillosis.

Objective To analyze the etiology of fever of unknown origin (FUO).Methods A total of 372 patients with FUO who hospitalized in Capital Medical University Affiliated Beijing Friendship Hospital were retrospectively analyzed from January 2003 to August 2013.All the patients were divided into two groups:group A (January 2003-December 2007) and group B (January 2008-August 2013).Diagnosis rate,duration of hospitalization (days) and time to diagnosis between the two groups were artificially compared.Results Of the 372 FUO cases,336 were positively diagnosed with a diagnosis rate of 90.3％.Infectious diseases were still the primary causes of FUO (60.2％),including 72 cases (32.1％) of tuberculosis.Connective tissue diseases accounted for 12.9％ of the FUO cases,malignancies were 8.3％,and miscellaneous diseases were 8.9％.Yet thirty six patients (9.7％) could not be confirmed until they were discharged from hospital.The duration of fever in patients with malignancies was longer than that with infectious diseases [60.0 (30.0,90.0) days vs 30.0 (20.0,60.0) days,P =0.003].Time to diagnosis of connective tissue disease and malignancies was longer than infectious diseases [(12.0 (7.3,18.8) days and 11.0 (7.0,18.0) vs 5.0 (3.0,8.0) days,both P values =0.000].The duration of hospitalization in group A was longer than that of group B [17.0(12.0,30.0) days vs 14.0(10.0,20.0) days,P =0.000].The diagnosis rate and time to diagnosis of group A were similar with those of group B.The proportion of connective tissue diseases in group A was higher than group B(18.1％ vs 9.2％,x2 =6.201,P =0.013).The proportion of infectious disease,malignancies and miscellaneous diseases was not significantly different between the two groups.Conclusions Infectious diseases are the major causes of FUO,and the most common cause is tuberculosis.Connective tissue diseases and malignancies are the second and third causes of FUO.The duration of fever and time to diagnosis are significantly different between the different origins.

Objective To observe the application effect of Yunnan baiyao powder and alcohol with VitB12 on exosmosis of vincristin. Methods The 40 patients with exosmosis of vincristin were randomly divided into the treatment group(22 cases)and the control group( 18 cases).The treatment group was given the Yunnan baiyao powder and alcohol with VitB12,the control group was given magnesium sulfate for hydropathic compress.The effective rate and red swelling and ulcer of the skin in the two groups were evaluated. Results The effective rate of the treatment group was higher than that of the control group,red swelling and the diameter of the ulcer of the skin on the fifth and seventh day were shorter than the control group. Conclusions The Yunnan baiyao powder and alcohol with VitB12 are effective in treatment of the exosmosis induced by vincristin.