ObjectiveTo investigate the effects and mechanism of the combination of arsenic trioxide （ATO） and its dimethylarsinic acid （DMAV） metabolite on apoptosis of human acute promyelocytic leukemia NB4 cells by affecting the balance of metabolic reaction. MethodsThe rats were injected with the same amount of sterile normal saline， ATO（1.6 mg·kg^-1）， and DMAV（4， 8， 16， and 32 mg·kg^-1） combined with ATO（1.6 mg·kg^-1）， respectively， to obtain the corresponding drug-containing serum. The effect of drug-containing serum on the proliferation of NB4 cells was detected by the cell counting kit-8 （CCK-8 ）method. Apoptosis was detected by flow cytometry with Annexin-V-FITC/PI double staining. Intracellular reactive oxygen species （ROS） accumulation was detected by flow cytometry using fluorescent probe DCFH-DA. The changes of mitochondrial membrane potential （Δψm） were detected by the fluorescence probe JC-1. Western blot detected the expression of apoptosis-related proteins， namely B-cell lymphoma-2（Bcl-2）-associated X protein（Bax）/Bcl-2， Cytochrome C， cleaved Caspase-9， and cleaved Caspase-3， as well as c-Jun N-terminal kinase（JNK） phosphorylation levels. Results①The Combination of the two drugs had a higher proliferation inhibition rate and more apoptosis than ATO alone. ②The combination of two drugs can significantly increase the production of ROS compared with any single treatment group. ③The Δψm was significantly reduced in the two-drug combination group compared with any single treatment group. ④Compared with either group， the combination group significantly released Cytochrome C， significantly down-regulated the expression of Bcl-2， and up-regulated the expression of Bax， cleaved Caspase-3， and cleaved Caspase-9. ⑤The phosphorylation level of JNK was significantly up-regulated in the two-drug combination group compared with any single treatment group. ConclusionThe combination of DMAV and ATO may synergistically induce mitochondrial apoptosis through ROS-mediated oxidative stress， triggering Δψm dissipation and Cytochrome C release. By activating Caspase-9/Caspase-3 and the phosphorylation level of JNK， the Bcl-2 family protein （Bax/Bcl-2） was regulated to promote the apoptosis of NB4 cells.

Objective:To explore the impact of Cullin5 on the radiosensitivity of glioblastoma multiforme cell and its underlying mechanisms.Methods:Glioblastoma U251 cells were transfected with flag-NC plasmids and overexpression plasmids (flag-Cullin5), and divided into the flag-NC group and flag-Cullin5 group. The expression levels of Cullin5 and programmed death ligand 1 (PD-L1) mRNA and protein were assessed using real-time reverse transcription polymerase chain reaction (qRT-PCR) and Western blot. After exposing U251 cells to 0, 2, 4, and 8 Gy X-ray radiation, cell viability was evaluated via the CCK-8 assay, and apoptosis rates were measured using flow cytometry. Co-immunoprecipitation was used to detect the interaction between Cullin5 and PD-L1, and ubiquitination assays were conducted to investigate the ubiquitination effect of Cullin5 on PD-L1. Cells were further transfected with PD-L1 overexpression (oe-PD-L1) and flag-Cullin5 and assigned into the flag-Cullin5 group, flag-Cullin5+oe-NC group, and flag-Cullin5+oe-PD-L1 group. qRT-PCR and Western blot were used to measure the expression levels of Cullin5 and PD-L1 mRNA and protein post-X-ray radiation in these groups. Cell survival rates and apoptosis rates were measured following radiation. Statistical differences between two or among multiple groups were analyzed using LSD- t test or one-way ANOVA. Results:Compared with the flag-NC group, the flag-Cullin5 group showed increased expression levels of Cullin5 mRNA and protein, and decreased PD-L1 protein expression (all P<0.05). Following radiation doses of 4 and 8 Gy, there was a reduction in cell survival rates and an increase in apoptosis rates (both P<0.05). Co-immunoprecipitation assay revealed the presence of PD-L1 in the Cullin5 immunoprecipitate and vice versa. Ubiquitination levels of PD-L1 protein were higher in the flag-Cullin5 group compared to those in the flag-NC group. Additionally, compared to the flag-Cullin5 group, the flag-Cullin5+oe-PD-L1 group exhibited increased PD-L1 mRNA and protein levels (both P<0.05), and following radiation doses of 4 and 8 Gy, these cells showed higher survival rates and lower apoptosis rates (both P<0.05). Conclusion:Cullin5 enhances the radiosensitivity of glioblastoma multiforme cells to radiation therapy through mediating the ubiquitination degradation of PD-L1.

Objective To study the clinicopathological features of ovarian clear cell carcinoma to pro-vide a basis for the differential diagnosis of whether it originates from endometrioid cyst.Methods A total of 30 patients with ovarian clear cell carcinoma receiving treatment in Jinjiang Municipal Hospital from January 2009 to December 2022 were collected as the research subjects.The patients not originating from endometrioid cysts were included in the control group(n=15)and the patients originating from endometrioid cysts were in-cluded in the observation group(n=15).Macroexamination,microscopic examination,hematoxylin-eosin stai-ning and immunohistochemical staining were used to analyze the clinicopathological characteristics.The gener-al data and pathological examination results of the two groups were statistically analyzed.Results Among the two groups,there were 22 cases(73.33％)of clear cells,3 cases(10.00％)of shoe-tack like cells,2 cases(6.67％)with about half of each cell type,and 3 cases(10.00％)of hyaline bodies or eosinophils.There were 27 cases(90.00％)of aspartic acid peptidase A(Napsin-A)positive and 29 cases(96.67％)of hepatocyte nu-clear factor-1β(HNF-1β)positive.Staging,ascites,lymph node metastasis,abdominal/pelvic metastasis,CA125,complicating endometrioid cyst,coffee-colored fluid in the sac,uneven thickness of the cyst wall,men-strual disorders and infertility all were the influencing factors of ovarian clear cell carcinoma originating from endometrioid cyst(P＜0.05).Conclusion The relevant pathological features could provide an important ref-erence base for the clinical differential diagnosis of whether ovarian clear cell carcinoma originates from endo-metrioid cysts,and it is of great significance to improve the detection rate of ovarian clear cell carcinoma.

Objective To evaluate the quality of life of patients with type 2 diabetes mellitus in Weifang City, Shandong Province and to explore its influencing factors. Methods A multistage stratified random sampling method was used to investigate patients in endocrine outpatient clinics in four medical institutions in Weifang from July to September 2022. The survey included general information, multi-dimensional evaluation of quality of life with the EQ-5D-5L scale, calculation of health utility values, and analysis of influencing factors using Tobit regression models. Results A total of 397 patients with type 2 diabetes were included in the present investigation, with health utility value of 0.82±0.21 points and visual analogue scale (VAS) score of 79.47±12.81 points. Pain or discomfort, anxiety or depression were more prominent in the study population. Age, diabetic complications, BMI, daily need for care, social support, and daily level of glycemic control were factors influencing health-related quality of life in patients with type 2 diabetes. Conclusion In the actual treatment of type 2 diabetes patients, an emphasis should be placed on protecting elderly type 2 diabetic patients, preventing and controlling the occurrence and development of diabetic complications, and improving daily blood glucose control to further improve the health-related quality of life of the population.

Objective:In order to explore the impact of corona virus disease 2019(COVID-19)on the hospitalization of children with bronchiolitis and to improve clinicians′ understanding of the characteristics of bronchiolitis during the COVID-19 epidemic.Methods:This was a multicenter clinical study, and the data have been collected from 23 children′s medical centers in China.All the clinical data were retrospectively collected from children with bronchiolitis who were hospitalized at each study center from January 1, 2019 to December 31, 2021.The results included gender, age at hospitalization, length of stay, respiratory syncytial virus(RSV) test results, severity rating, ICU treatment, and the total number of children hospitalized with respiratory tract infection during the same period.The clinical data of children with bronchiolitis in 2019 before COVID-19 epidemic and in 2020、2021 during COVID-19 epidemic were statistically analyzed and compared.Results:According to a summary of data provided by 23 children′s medical centers, there were 4 909 cases of bronchiolitis in 2019, 2 654 cases in 2020, and 3 500 cases in 2021.Compared with 2019, the number of bronchiolitis cases decreased by 45.94% in 2020 and 28.70% in 2021.In 2019, 2020 and 2021, there were no significant differences in gender ratio, age, and duration of hospitalization.Compared with 2019, the ratio of bronchiolitis to the total number of hospitalizations for respiratory tract infection decreased significantly in 2020 and 2021( χ2=12.762, P<0.05; χ2=84.845, P<0.05).The proportion of moderate to severe bronchiolitis cases in both 2020 and 2021 was lower than that in 2019, and the difference was statistically significant ( χ2=4.054, P<0.05; χ2=8.109, P<0.05).There was no statistically significant difference in the proportion of bronchiolitis cases requiring ICU treatment between 2019, 2020, and 2021 ( χ2=1.914, P>0.05).In 2019, a total of 52.60%(2 582/4 909) of children with bronchiolitis underwent RSV pathogen testing, and among them, there were 708 cases with RSV positive, accounting for 28.00%.In 2020, 54.14%(1 437/2 654) of children with bronchiolitis underwent RSV pathogen testing, and there were 403 cases with RSV positive, accounting for 28.04%.In 2021, 66.80%(2 238/3 500) of children with bronchiolitis underwent RSV pathogen testing, and there were 935 cases with RSV positive, accounting for 41.78%.Compared with 2019 and 2020, the RSV positive rate in 2021 showed a significant increase( χ2=99.673, P<0.05; χ2=71.292, P<0.05). Conclusion:During the COVID-19 epidemic, the implementation of epidemic prevention and control measures reduced the hospitalization rate and severity of bronchiolitis, but did not reduce the positive rate of RSV detection.

Objective To analyze the etiological characteristics and the variation of pathogens spectrum in hospitalized children with a-cute respiratory tract infection(ARTI)before and after COVID-19 under"the level B of management for class B"of infectious diseases(Level B for Class B)in Shenzhen,in order to provide reference for the clinical diagnosis,treatment and prevention of ARTI.Meth-ods The ARTI cases from January 8,2022 to July 30,2022 were selected as before"Level B for Class B",and the cases from Janu-ary 8,2023 to July 30,2023 were selected as after"Level B for Class B".The pharyngeal swab samples submitted for analyzing 11 common pathogens,such as COVID-19,influenza virus(Ⅳ),respiratory syncytial virus(RSV)and mycoplasma pneumoniae(MP)in the children with ARTI admitted to Shenzhen Children's Hospital.Results SARS-CoV-2 were detected as positive in 347 cases,a-mong which 225 cases were before"Level B for Class B"including 29 cases combined with other pathogens(12.89％,29/225)and human parainfluenza viruses(HPIV)was the most common(31.03％,9/29).After"Level B for Class B",SARS-CoV-2 were detec-ted as positive in 122 cases,including 28 cases combined with other pathogens(22.95％,28/122),and RSV was the most common(28.57％,8/28).There was a statistical difference between the positive rate of SARS-CoV-2 combined with other pathogens before and after"Level B for Class B"(X2=5.834,P=0.016).After"Level B for Class B",the total pathogen detection rate(positive for at least one pathogen)was 60.82％(2 864/4 709)in the spring(January 8,2023 to April 30,2023),and influenza virus A(IVA)(22.64％,1 066/4 709),rhinovirus(HRV)(19.86％,935/4 709)and RSV(13.29％,626/4 709)were the main pathogens,and there were 301 cases(6.39％,301/4 709)of mixed infections.In the summer(May 1,2023 to July 30,2023),the total detection rate of pathogens was 70.26％(4 012/5 710),among which RSV(21.63％,1 235/5 710),MP(13.91％,794/5 710),HPIV(10.05％,574/5 710)were the main pathogens,and there were 710 cases(12.43％,710/5 710)of mixed infections,all of which were significantly higher than the same period before"Level B for Class B".The difference was statistically significant(P＜0.05).Conclusion After"Level B for Class B"for COVID-19,the detection rate of 11 common pathogens increased significantly and the pathogen spectrum of ARTI changed significantly.

Traumatic cerebrospinal fluid leakage commonly presents in traumatic brain injury patients, and it may lead to complications such as meningitis, ventriculitis, brain abscess, subdural hematoma or tension pneumocephalus. When misdiagnosed or inappropriately treated, traumatic cerebrospinal fluid leakage may result in severe complications and may be life-threatening. Some traumatic cerebrospinal fluid leakage has concealed manifestations and is prone to misdiagnosis. Due to different sites and mechanisms of trauma and degree of cerebrospinal fluid leak, treatments for traumatic cerebrospinal fluid leakage varies greatly. Hence, the Craniocerebral Trauma Professional Group of Neurosurgery Branch of Chinese Medical Association and the Neurological Injury Professional Group of Trauma Branch of Chinese Medical Association organized relevant experts to formulate the " Chinese expert consensus on the diagnosis and treatment of traumatic cerebrospinal fluid leakage in adults ( version 2023)" based on existing clinical evidence and experience. The consensus consisted of 16 recommendations, covering the leakage diagnosis, localization, treatments, and intracranial infection prevention, so as to standardize the diagnosis and treatment of traumatic cerebrospinal fluid leakage and improve the overall prognosis of the patients.

Objective:To effectively express the receptor binding domain (RBD) of SARS-CoV-2 spike protein in Pichia pastoris and to evaluate its immunogenicity. Methods:The gene encoding the RBD protein was synthesized and cloned into the pPICZαA plasmid. After linearization, the plasmid was transferred and integrated into the genome of Pichia pastoris. The expressed RBD protein in culture supernatant was analyzed by Western blot and Biolayer interferometry. After screening, a single clone expressing the RBD protein was selected. The high-level expression of RBD protein was achieved by optimizing the fermentation process, including the salt concentration adjusting of the medium and induction condition optimization (pH, temperature and duration). The immunogenicity of the expressed RBD protein was evaluated in a mouse model. Results:A single clone with a high expression level of RBD protein was obtained and named RBD-X33. The expression level of RBD protein in the fermentation supernatant reached up to 240 mg/L after optimization of the induction condition (HBSM medium, pH=6.5±0.3, 22℃ and 120 h). In the mouse experiment, the recombinant RBD protein was formulated with Alum+ CpG dual adjuvant and injected into mice. The binding IgG antibody levels were up to 2.7×10 6 tested by ELISA and the neutralizing antibody levels were up to 726.8 tested by live virus neutralizing antibody assay (prototype). Conclusions:The RBD protein could be efficiently expressed in Pichia pastoris and induce stronger immune response in animals. This study suggested that the recombinant SARS-CoV-2 RBD protein expressed in Pichia pastoris could serve as a candidate antigen in the development of SARS-CoV-2 vaccine.

Objective:To explore the correlation between the expression of signaling lymphocyte activation molecule family 6 (SLAMF6) on peripheral blood CD8 +T cells and perforin and granzyme B and the clinical significance in patients with newly diagnosed severe aplastic anemia(SAA). Methods:The indicators of blood routine and bone marrow and peripheral blood samples of 32 newly diagnosed SAA patients admitted to Henan Provincial People′s Hospital from January 2016 to June 2019 were collected for retrospective analysis. Flow cytometry was used to detect the expression of SLAMF6, perforin and granzyme B on samples CD8 +T cell before therapy and 6 months after therapy (11 cases received transplantation, 21 cases received immunosuppressive therapy [IST]). Spearman correlation analysis was performed to determine the association between clinical indicators and laboratory test results. The expression of SLAMF6, perforin and granzyme B was also detected in 10 healthy people (normal group) and 13 myelodysplastic syndromes/paroxysmal nocturnal hemoglobinuria (MDS/PNH) patients (MDS/PNH group). Results:(1) At diagnosis: the expression of SLAMF6 was significantly lower in the SAA group than that in the normal group and the MDS/PNH group ([56.40±6.37]% vs [84.34±5.81]% and [82.24±4.98]% (both P<0.001]). The expression of perforin was significantly higher in the SAA group (32.73±8.46) than that in the normal control group (23.75%±5.10%), and the MDS/PNH group (26.12%±5.53%) (both P<0.05). The expression of granzyme B was also significantly higher in the SAA group (36.23%±7.94%) than that in the normal control group (21.67%±5.05%) and the MDS/PNH group (21.79%±5.10%) (both P<0.001). The expression of SLAMF6 was positively correlated with the hemoglobin ( r=0.804), and reticulocyte absolute values ( r=0.656) in peripheral blood, percentage of granulocytes ( r=0.643) and erythrocytes ( r=0.622) in bone marrow of SAA patients (all P<0.05). Expression of SLAMF6 was negatively correlated with perforin ( r=-0.792) and granzyme B ( r=-0.908) on CD8 +T cells in patients with SAA (both P<0.001). (2) After treatment: the expression of SLAMF6 in peripheral blood CD8 +T cells of 30 surviving patients was higher than pre-treatment ([79.19±12.69]% vs [56.40±6.37]%, P<0.001). The expressions of perforin and granzyme B were lower than pre-treatment level (both P<0.05). The expression of SLAMF6 on CD8 +T cells in 11 transplanted patients was higher than before transplantation ([86.54±3.75]% vs [56.40±7.35]%, P<0.001). The expressions of perforin and granzyme B were lower than before transplantation (both P<0.05). The expression of SLAMF6 on CD8 +T cells in 12 IST-respond patients was higher than that before treatment, while the perforin and granzyme B levels were lower than pre-treatment (all P<0.05). The post-treatment expressions of SLAMF6, perforin and granzyme B were similar as before treatment levels in 7 IST-unrespond patients (all P>0.05). Conclusion:SLAMF6 is significantly down-regulated on CD8 +T cells in newly diagnosed SAA, negatively correlated with the effective factors of CD8 +T cells, which might participate in the immune regulatory of CD8 +T cells as a negative regulatory factor in patients with SAA. The SLAMF6 is significantly up-regulated after hematopoietic recovery, while there is no significant change in treatment-unrespond patients, which could thus serve as an useful diagnostic and therapeutic index of patients with SAA.

ObjectiveTo investigate the value of peroxisome proliferator-activated receptor γ（PPARγ）level in predicting hemorrhagic transformation in patients with non thrombolytic acute cerebral infarction. MethodsA total of 92 patients with non thrombolytic acute cerebral infarction who were treated in our hospital from May 2017 to May 2020 were selected as the research objects，and they were divided into hemorrhagic transformation group（n=14）and non hemorrhagic transformation group（n=78）according to the occurrence of hemorrhagic transformation. The plasma levels of PPARγ and fibronectin（FN）were detected by enzyme-linked immunosorbent assay（ELISA）；the correlation between plasma PPAR-γand blood lipid levels in patients with hemorrhagic transformation of non thrombolytic acute cerebral infarction was analyzed；the predictive value of plasma PPARγ and FN levels on hemorrhagic transformation in patients with non thrombolytic acute cerebral infarction was analyzed；the influencing factors of hemorrhagic transformation in patients with non thrombolytic acute cerebral infarction were analyzed. ResultsThe levels of PPARγ and FN were higher than those in non hemorrhagic transformation group，The area under the curve（AUC）of plasma PPARγ and FN levels in predicting hemorrhagic transformation in patients with non thrombolytic acute cerebral infarction was 0.933 and 0.860，respectively，the specificity was 89.7% and 78.2%，and sensitivity was 85.7% and 78.6%，respectively；the AUC of the combined prediction was 0.952，the specificity was 89.7%，and the sensitivity was 92.9%. PPARγ was an independent risk factor for hemorrhagic transformation in patients with non thrombolytic acute cerebral infarction（P<0.05）. ConclusionPlasma PPARγ is a factor influencing hemorrhagic transformation in patients with non thrombolytic acute cerebral infarction，which may have important predictive value for secondary hemorrhagic transformation.