Background::Hepatic ischemia-reperfusion injury (HIRI) remains a common complication during liver transplantation (LT) in patients. As a key downstream effector of the Hippo pathway, Yes-associated protein (YAP) has been reported to be involved in various physiological and pathological processes. However, it remains elusive whether and how YAP may control autophagy activation during ischemia-reperfusion.Methods::Human liver tissues from patients who had undergone LT were obtained to evaluate the correlation between YAP and autophagy activation. Both an in vitro hepatocyte cell line and in vivo liver-specific YAP knockdown mice were used to establish the hepatic ischemia-reperfusion models to determine the role of YAP in the activation of autophagy and the mechanism of regulation. Results::Autophagy was activated in the post-perfusion liver grafts during LT in patients, and the expression of YAP positively correlated with the autophagic level of hepatocytes. Liver-specific knockdown of YAP inhibited hepatocytes autophagy upon hypoxia-reoxygenation and HIRI ( P <0.05). YAP deficiency aggravated HIRI by promoting the apoptosis of hepatocytes both in the in vitro and in vivo models ( P <0.05). Attenuated HIRI by overexpression of YAP was diminished after the inhibition of autophagy with 3-methyladenine. In addition, inhibiting autophagy activation by YAP knockdown exacerbated mitochondrial damage through increasing reactive oxygen species ( P <0.05). Moreover, the regulation of autophagy by YAP during HIRI was mediated by AP1 (c-Jun) N-terminal kinase (JNK) signaling through binding to the transcriptional enhanced associate domain (TEAD). Conclusions::YAP protects against HIRI by inducing autophagy via JNK signaling that suppresses the apoptosis of hepatocytes. Targeting Hippo (YAP)-JNK-autophagy axis may provide a novel strategy for the prevention and treatment of HIRI.

This study aims to obtain nanoantibody of vaccinia virus protein D8 by SUMO tag and analyze its binding activity with antigen.The sequence was designed and synthesized to construct the prokaryotic expression plasmid pKMD-SUMO-D8,which was transformed into Escherichia coli BL21(DE3)receptor cells and induced to express.Anti-SUMO-D8 with His and SUMO tags were purified,and then all tags were removed by SUMO protease to further obtain Anti-D8.Final-ly,the binding activity was detected and analyzed by indirect ELISA and IFA.The results showed that the prokaryotic expression plasmid pKMD-SUMO-D8 and recombinant strains were construc-ted.Under the condition of 30 ℃,the expression of Anti-SUMO-D8 was best when induced by IPTG with 0.1 mmol/L for 4 h.High purity Anti-SUMO-D8 and Anti-D8 nanoantibodies were ob-tained.Indirect ELISA results showed that both Anti-SUMO-D8 and Anti-D8 had binding activity with antigen.Under the same conditions,the binding activity of Anti-D8 to vaccinia virus and E8L was higher than that of Anti-SUMO-D8.Indirect IFA results showed that 48 h after infection with vaccinia virus,some nuclei of the experimental group showed green fluorescence under fluorescence microscope.Nanoantibodies of vaccinia virus protein D8 could be efficiently prepared by SUMO tag,and the prepared nanoantibodies had binding activity.This study provides new idea for the preparation of nanoantibody,and also lays a foundation for further study of the biological function of vaccinia virus protein D8 nano-antibody.

Objective:To investigate the antioxidant function of Mycoplasma pneumoniae MPN662 and analyze the key active sites, and to explore the role of MPN662 in the regulation of the production of reactive oxygen species (ROS) and superoxide dismutase (SOD) in THP-1 cells. Methods:pET28a(+ )- mpn662, recombinant mutant plasmids pET28a(+ )- mpn662-Ser 66 (the 66 th Cys was mutated to Ser) and pET28a(+ )- mpn662-Ala 66 (the 66 th Cys was mutated to Ala) were constructed, recombinant proteins rMPN662, rMPN662-Ser 66 and rMPN662-Ala 66 were expressed, identified, and purified. DTNB method was employed to analyze the MetO reduction activity of rMPN662 and recombinant mutant protein. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot were applied to examine the transcription level of the mpn662 gene and the expression level of MPN662 protein after Mycoplasma pneumoniae were stimulated with different concentrations of hydrogen peroxide (H 2O 2) or tert-butyl hydroperoxide (t-BHP), respectively. Fluorescent probes (DCFH-DA) and the total SOD activity detection kit were used to test the levels of intracellular ROS and SOD in THP-1 cells, which were pretreated with rMPN662, and then stimulated by Mycoplasma pneumoniae lipid-associated membrane proteins (LAMPs). Results:Mycoplasma pneumoniae rMPN662 could reduce MetO to Met, and the enzyme activities of mutant protein were significantly lower than those of rMPN662 protein. mpn662 gene mRNA transcription level and MPN662 protein expression level were significantly increased in a dose-dependent manner when Mycoplasma pneumoniae was stimulated with H 2O 2 and t-BHP. Treatment with rMPN662 before THP-1 cells were exposed to LAMPs could decrease the level of ROS and increase the production of SOD. Conclusions:Mycoplasma pneumoniae MPN662 can reduce MetO to Met, and Cys66 is the key amino acid for this activity. MPN662 can decrease the release of ROS and increase the production of SOD in Mycoplasma pneumoniae LAMPs stimulated THP-1 cells.

Abstract: Objective To develop an ELISA kit to detect IgG antibodies of Chikungunya virus (CHIKV), providing a new method for epidemiological investigation and detection in the field for CHIKV infection. Methods Using the CHIKV-specific recombinant protein pMal-chik23 as diagnostic antigen, HRP-labeled anti-IgG antibody as color-developing antibody, and the working concentration of diagnostic antigen, serum to be tested and second antibody were optimized using orthogonal. The reaction conditions of ELISA reaction, such as coating, blocking, incubation, and color-developing were systematically optimized. The cut-off value for ELISA detection was established based on the assessment of a large clinical sample set. On this basis, the specificity, sensitivity, and stability of the ELISA response were evaluated to develop and assemble a rapid ELISA kit for the detection of Chikungunya fever IgG antibodies. Results On the basis of systematic conditions optimization, an indirect ELISA kit for the detection of IgG antibodies against CHIKV was developed and assembled. The optimal reaction conditions were identified as 1.0 μg/mL antigen was coated using carbonate buffer at 4 ℃ for 24 hours. Then the microplate was blocked using HBV blocking solution at 37 ℃ for 4 hours. 100 μL/well samples to be tested were diluted at 1∶101, reacted at 37 ℃ for 40 minutes, and washed 4 times with PBST. Thus, HRP-labeled rabbit anti-human IgG was diluted at 1∶20 000, HRP-labeled sheep anti-mouse IgG was diluted at 1∶10 000, reaction at 37 ℃ for 30 minutes, and washed 5 times with PBST. Finally, 100 μL/well TMB solution was added and incubated at 37 ℃ for 10 minutes. Then terminate the reaction with 50 μL of 20% H2SO4 and measure the A450 value at dual wavelengths of 450/630 nm (A450) . The evaluation results showed that ELISA A450 of Chikungunya fever-positive samples were more than 0.43, while the ELISA A450 of negative samples was less than 0.04, and the S/N ratio > 10. Specificity test showed that the developed kit had no cross-reaction with 9 other similar arbovirus species such as Sindbis, Geta, Ross River, and Dengue virus. The stability evaluation of the reagent kit indicated that it had high stability, with a coefficient of variation (CV) within the microplate ranging from 0.76% to 2.12%, the coefficient of variation between the microplate ranged from 0.64% to 1.85%, and the coefficient of variation between batches ranged from 0.83% to 2.31%, all of which were less than 3%. The sensitivity of the kit did not decrease significantly after being stored at 4°C for 1 year. Conclusions A rapid indirect ELISA kit for the detection of Chikungunya fever IgG antibodies was successfully developed, exhibiting good sensitivity, specificity, and stability.

To address the challenges of detecting tuberculosis pathogens in sputum smear images,such as complex backgrounds,small targets,and high costs of manual screening,a detection method based on YOLOv8s is presented.The structure is improved through spatial and channel reconstruction convolutions to limit feature redundancy,and a coordinate attention is introduced to expand the receptive field of the model.Furthermore,a spatial pyramid pooling cross-stage partial network is used to extract feature information at different scales,and a normalized attention mechanism is embedded to suppress less significant features.The experimental results on a public dataset show that compared with the original YOLOv8s model,the improved network model enhances precision and recall rates by 2.7%and 1.5%,respectively,and improved mean average precision at a confidence level of 0.5 by 2.3%,demonstrating that the improved model can effectively assist radiologists in diagnosis.

Objective To analyze and compare the clinical efficacy of CalliSpheres drug-eluting micro-spheres and blank microspheres in the treatment of advanced non-small cell lung cancer by bronchial arterial chemoembolization.Methods Fifty patients with advanced non-small cell lung cancer who had failed or relapsed after radiotherapy,chemotherapy,targeting and immunotherapy were collected and treated with super-selective bronchial artery chemoembolization.A retrospective analysis was conducted to compare the tumor response rate and survival between CalliSpheres drug-eluting and blank microspheres.Results The PR,ORR and DCR in the drug-eluted microsphere group were higher than those in the blank microsphere group,and there was a statistical difference in DCR between the two groups 1 month after surgery(χ2 = 4.08,P = 0.04).PD in the drug-eluted microsphere group was lower than that in the blank microsphere group.The CEA,CYF and SCC in the drug-eluted microsphere group after surgery were lower than those in the blank microsphere group,and the CEA,CYF and SCC in the two groups after surgery were lower than those before surgery,and there were statistical differences in CEA and CYF 1 month after surgery between the two groups.The PFS and OS in drug-eluted microsphere group were higher than those in blank microsphere group.Conclusion CalliSpheres drug-eluting microspheres could improve the effective rate of tumor treatment and prolong the survival time more effectively than the blank micro-spheres via arterial chemoembolization,providing reliable clinical practice basis for the treatment of advanced non-small cell lung cancer.

Objective:Clinical characteristics and diagnosis and treatment process was reported and analyzed of a patient with brucellosis complicated with thyroid abscess, providing reference for the clinical diagnosis of brucellosis complicated with thyroid abscess.Methods:Clinical medical records of a patient with brucellosis complicated with thyroid abscess who was treated at the General Surgery Department of Yanchi County People's Hospital in Wuzhong City, Ningxia Hui Autonomous Region in November 2021 were collected. The clinical manifestations, blood routine, brucella antibodies, thyroid function, bacterial culture, thyroid ultrasound and other examination results, as well as the diagnosis and treatment process, were comprehensively analyzed. Results:The patient was a male, 61 years old, who presented with a neck mass without typical clinical manifestations of brucellosis. Thyroid ultrasound revealed a space occupying lesion, and the preliminary diagnosis was thyroid cystadenoma. Thyroid right lobe and isthmus resection surgery was performed. During the operation, it was found that some of the thyroid glands were tightly adhered to the cervical blood vessels, so the resection surgery was changed to abscess drainage, and the drainage fluid was purulent and bloody. The bacterial culture result of thyroid purulent fluid (intraoperative puncture fluid and postoperative drainage fluid) was brucella lamblia, and the serum brucella test tube agglutination test titer was 1 ∶ 400 (+++). The patient improved and was discharged after local drainage and anti brucella treatment. Follow up for 4 months showed no abnormalities. Conclusions:Brucellosis which begins with a local infection of the thyroid gland is extremely rare, with no characteristic clinical manifestations, and is prone to misdiagnosis. Timely correction of the surgical plan during the treatment process avoids the removal of the patient's thyroid, which has a certain clinical reference value.

Objective:To explore the relationship between the neural development of preterm infants and gut microbiota.Methods:66 premature infants who were hospitalized in the Neonatology Department of Hunan Children′s Hospital from September 2018 to September 2019 were included in the study. Their fecal samples and clinical data from the first admission were collected. According to the neurodevelopment, the patients were divided into normal neurodevelopment group and neurodysplasia group. The bacterial DNA of fecal samples was extracted by 16S rDNA high-throughput sequencing technology and bioinformatics analysis was conducted to compare the composition and diversity of gut microbiota between the two groups.Results:(1) The Shannon index of gut microbiota in normal neurodevelopmental group and neurodysplastic group was 0.89(0.41, 1.51) and 1.01(0.47, 1.31), respectively. There was no significant difference in diversity index between the two groups ( P>0.05). (2) Bifidobacterium, veronica and negativites in the gut microbiota of the normal neurodevelopmental group were significantly higher (all P<0.05), and streptococcus in the gut microbiota of the dysplastic group were significantly higher ( P<0.05). The gut microbiota of the two groups were mainly enterococcus and escherichia shigella. Conclusions:At the genus level, enterococcus and escherichia are the dominant flora of early gut microbiota in preterm infants. Gut microbiota is related to the neural development of preterm infants. The increased abundance of streptococcus, and the decreased abundance of bifidobacterium, veronicus, and negativites may be risk factors for neurodysplasia of preterm infants. The diversity of gut microbiota in early preterm infants may not be significantly related to neural development.

Objective To explore the effect of intervention model based on the framework of in-tervention mapping theory on psychiatric symptoms and impulsive behavior of male schizophrenic pa-tients.Methods A total of 80 patients with schizophrenia were selected as study objects.According to the random number table method,the patients were randomly divided into control group and obser-vation group,with 40 cases in each group.The control group was given routine psychiatric intervention model,and the observation group was given intervention model based on intervention mapping theoreti-cal framework on the basis of routine intervention.The insight status[Insight and Treatment Attitude Questionnaire(ITAQ)],psychiatric symptoms[Positive and Negative Syndrome Scale(PANSS)],psychological status[Connor-Davidson Resilience Scale(CD-RISC)and Self-esteem Scale(SES)]and impulsive behavior[Nurses Observation Scale for Inpatients(NOSIE)]were compared between the two groups.Results After intervention,the total score of ITAQ and the score of each dimension in the two groups increased,and were higher in the observation group than those in the control group(P＜0.05).The total score of PANSS and the score of each dimension of the two groups decreased,and were lower in the observation group than those in the control group(P＜0.05).After interven-tion,the scores of CD-RISC and SES in the two groups increased(P＜0.05).The scores of NOSIE and the incidence of impulsive behavior in the observation group were lower than those in the control group(P＜0.05).Conclusion The intervention model based on the intervention mapping theory framework can improve the insight state of male schizophrenic patients,improve their mental symp-toms and psychological state,and effectively reduce the incidence of impulsive behavior.

Objective:To explore the potential genetic causes of unexplained neonatal encephalopathy.Methods:This retrospective study enrolled 113 infants diagnosed with unexplained neonatal encephalopathy and underwent genetic testing in the Children's Hospital of Hunan Province from January 2019 to May 2021. Perinatal data, clinical manifestations, electroencephalograph, brain MRI findings, genetic information, and prognosis of those patients were analyzed. T-test or Chi-square test were used for data analysis. Results:Of the 113 infants enrolled, 74 (65.5%) were males. The gestational age at birth was (38.6±1.5) weeks, and the birth weight was (2 957±561) g. The most common clinical manifestation was the disturbance of consciousness (83/113, 73.5%), followed by seizures (39/113, 34.5%). There were 38.2% (34/89) of the patients with abnormal brain MRI, and 80.4% (74/92) presented abnormal electroencephalography. Among the 113 infants, 60 (53.1%) had genetic abnormalities, including 48 with single nucleotide variations, eight with copy number variations, and four with chromosome abnormalities. Single nucleotide variations in the 48 patients were classified into syndromic ( n=18, 37.5%), metabolic ( n=16, 33.3%), epileptic ( n=11, 22.9%) and mitochondrial-related genes ( n=3, 6.3%), of which 14 were not included in any database. Among the 103 cases which were successfully followed up until December 31, 2021, 75 (72.8%) had a poor prognosis, including 52 (50.5%) death cases and 23 (22.3%) cases of development retardation. Birth weight and the incidence of seizures in the poor prognosis group were both lower than those in the non-poor prognosis group [(2 876±536) vs (3 254±554) g, t=3.15; 29.3% (22/75) vs 53.6% (15/28), χ2=5.20; both P<0.05], while the incidence of disturbance of consciousness was higher [80.0% (60/75) vs 53.6% (15/28), χ2=7.19, P<0.05]. The proportion of infants with genetic abnormalities in the poor prognosis group was higher than that in the non-poor prognosis group, but the difference was not statistically significant [53.3% (40/75) vs 46.4% (13/28), χ2=0.39, P=0.533]. Conclusions:Genetic abnormality is one of the leading causes of unexplained neonatal encephalopathy. Nucleotide variation is the most common genetic type. Syndromic, metabolic, and epileptic variants are frequently detected in these patients.