Objective To prepare humanized monoclonal antibodies(Mabs)targeting Acinetobacter baumannii(Ab)based on single memory B cell sequencing technology,construct the immune repertoire of the core protein of Ab,hemolysin-coregulated protein(Hcp),and express its Mabs with binding activity.Methods E.coli BL21 harboring the recombinant plasmid pGEX-6p-1-Hcp was constructed.Hcp protein was obtained using protein expression and affinity chromatography.Female SPF BALB/c mice(6～8 weeks old,weighing 18～20 g)were immunized intramuscularly with antigen Hcp to generate specific memory B cells.Single antigen-specific memory B cells were sorted using flow cytometry.The immune repertoire of Hcp was constructed using single-cell sequencing technology,and bioinformatics analysis was performed on the sequencing results.Mabs were obtained using antibody humanization techniques.The in vitro binding activity of the antibodies was detected by ELISA.Results The target protein Hcp with a purity>95%was obtained after expression and purification.The immune repertoire of Hcp was successfully constructed,and the results of BCR clonotype identification and analysis,CDR3 region characteristic analysis,and V-J gene pairing characteristic analysis were achieved.Antibody humanization got 7 Mabs,that is,IgG1-1,IgG1-2,IgG2-1,IgG2-2,IgG3-1,IgG4-1 and IgG4-2.ELISA results showed IgG1-1,IgG3-1,IgG4-1,and IgG4-2 had an antibody binding titer of 1∶1 280,IgG2-2 of 1∶10 240,IgG2-1 of 1∶5 120,and IgG1-2 of 1∶160.Conclusion Single-cell sequencing technology enables rapid,accurate,and efficient construction of an Hcp protein immune repertoire containing extensive antibody information.Utilizing this immune repertoire allows for the expression of Mabs with binding activity.

Purpose/Significance To classify the elderly according to their heterogeneous health status,and to explore the potential categories and influencing factors status,so as to promote the precision of health information services for the elderly.Method/Process Based on the data of the China health and retirement longitudinal study(CHARLS)database in 2018,the elderly are classified according to their health status by the method of latent class analysis,and the main influencing factors are identified by regression analysis.Result/Conclusion The elderly could be divided into 4 categories according to their health status.Age,sex,education level and retirement sta-tus are significant factors affecting the health grouping of the elderly.According to the heterogeneous health characteristics of the elderly,the service optimization strategy should be provided to promote the physical and mental health of the elderly.

Objective:To analyze the clinical characteristics of patients with spontaneous splenorenal shunt (SSRS) in liver cirrhosis, and to compare the effects and prognosis of different treatments.Methods:The data of cirrhotic patients with SSRS at the First Affiliated Hospital of Zhengzhou University between 2016-2022 were retrospectively analyzed.Patients were divided into Group A receiving conservative treatment, Group B by simple embolization, Group C undergoing TIPS combined with embolization, and Group D given liver transplantation. Life status, liver function changes, incidences of adverse events, and survival between groups were compared.Results:SSRS diameter was positively correlated with blood ammonia ( R=0.478) and negatively correlated with portal vein diameter ( R=-0.301). SSRS diameter is a protective factor for gastrointestinal hemorrhage and ascites and a risk factor for hepatic encephalopathy; Blood ammonia decreased and prothrombin time prolonged after treatment in group A ( P<0.05), blood ammonia decreased and albumin increased in group B ( P<0.05). Hemoglobin and bilirubin increased in group C ( P<0.05), blood ammonia and bilirubin decreased and platelets and albumin increased in group D ( P<0.05); Survival analysis showed that the prognosis of groups A and C was related to liver function, and the survival rate of group D was the highest of all ( P<0.05). Conclusions:SSRS embolization is safe and effective, and liver transplantation improves patient survival. Individualized treatment should be selected based on patient symptoms, liver function, and shunt diameter.

Oral health is closely related to systemic health. Common chronic oral diseases, periodontitis and periapical inflammation for example, not only affect the health of oral soft and hard tissues including the alveolar bone and gums, but may also cause changes in systemic conditions such as chronic low-grade inflammation, elevated oxidative stress levels, and dysbiosis of the microbiota. These changes in systemic health can exacerbate the progression of obesity. Therefore, through proactive oral health interventions such as maintaining good oral hygiene habits, modifying dietary structures, and undergoing oral examinations, it is possible to effectively prevent and alleviate inflammatory oral diseases, and actively intervene in obesity. This article delves into the impact of inflammatory oral diseases on obesity and their underlying mechanisms, defines the concept of "oral proactive healthcare", and systematically summarizes their preventive and therapeutic effects on inflammatory oral diseases, thereby demonstrating the potential of improving obesity through proactive oral health strategies.

To analyze the effects of tooth brushing frequency on blood indicators among adolescents and to preliminarily explore the effects of tooth brushing frequency on general health.

The Chinese Neonatal Network(CHNN) was established in 2018 with the mission of establishing a national collaboration platform, conducting high-quality and collaborative research, and ultimately improving the quality of neonatal-perinatal care and health in China.At present, 112 hospitals across the country have joined CHNN.CHNN has established a national standardized cohort of very premature infants/very low birth weight infants with >10 000 enrollments each year, has been leading data-driven collaborative quality improvement initiatives, conducting multicenter clinical studies, and performing multi-level training programs.Guided by the principles of collaboration and sharing, data-driven, continuous improvement, and international integration, CHNN has become an important platform for clinical and research collaboration in neonatal medicine in China.

Objective:To explore the predictive value of amplitude integrated electroencephalography(aEEG)in the neurological prognosis of children with neonatal bacterial meningitis(NBM).Methods:The clinical data and aEEG results from 148 children diagnosed with NBM who completed aEEG examinations in the Department of Neonatology at Kunming Children′s Hospital from January 2018 to December 2019 were retrospectively analyzed.According to whether aEEG is abnormal, the children were divided into aEEG abnormal group and aEEG non-abnormal group.According to the degree of aEEG abnormality, children with aEEG abnormality were divided into aEEG mild abnormal group and aEEG severe abnormal group.The abnormal rate and abnormal characteristics of aEEG were analyzed; The clinical data of two groups were compared.Results:(1)Among the 148 children with NBM, 49 children had abnormal aEEG, 99 children had no abnormality, and the aEEG abnormal rate was 33.1%.The abnormal aEEG was manifested as delayed sleep-wake cycle maturation in 39 (26.3%) cases, abnormal discharge in eight (5.4%) cases, and abnormal background activity in one (0.6%) case.(2)The proportion of children with convulsive seizures and refractory NBM in aEEG abnormal group were significantly higher than those in aEEG non-abnormal group ( P<0.05). In the routine and biochemical abnormal indexes of cerebrospinal fluid, the proportion of protein >3 g/L, cerebrospinal fluid leukocyte>500×10 6/L, cerebrospinal fluid glucose<1.5 mmol/L, positive cerebrospinal fluid culture, positive blood and cerebrospinal fluid culture, abnormal head MRI in aEEG abnormal group significantly increased ( P<0.05); While there was no significant difference regarding blood routine leukocyte abnormality, CRP increase, and positive blood culture ratio between two groups ( P>0.05). (3) 148 cases of NBM children were followed up to 15 months old, 119 (80.4%) cases completed the follow-up, the loss rate was 19.6%, three cases died, and 11 cases had psychomotor retardation.Compared with the children with abnormal aEEG, the prognosis of children with NBM was significantly different, the Spearman rank correlation coefficient r was 0.315 ( P<0.05). COX regression was used to analyze the predictive value of each index for adverse outcomes. Abnormal aEEG was an independent risk factor for adverse outcomes in children with NBM ( OR=7.452, 95% CI 1.605-34.591, P<0.05). Conclusion:The aEEG monitoring of children with NBM, if abnormal, may indicate severe NBM, which is likely to be transformed into refractory NBM or has a poor prognosis.

Streptococcus pyogenes Cas9 (SpCas9) has become a powerful genome editing tool, but has a limited range of recognizable protospacer adjacent motifs (PAMs) and shows off-target effects. To address these issues, we present a rational approach to optimize the xCas9 mutant derived from SpCas9 by directed evolution. Firstly, energy minimization with the Rosetta program was applied to optimize the three-dimensional structure of Cas9 to obtain the lowest energy conformation. Subsequently, combinatorial mutations were designed based on the mutations sites of xCas9 acquired during the directed evolution. Finally, optimal mutants were selected from the designed mutants by free energy ranking and subjected to experimental verification. A new mutant yCas9 (262A/324R/409N/480K/543D/694L/1219T) with multiple PAM recognition ability and low off-target effects was obtained and verified by DNA cleavage experiments. This mutant recognizes the NG, GAA and GAT PAMs and shows low off-target DNA cleavage activity guided by mismatched sgRNA, thus provides a gene editing tool with potential applications in biomedical field. Furthermore, we performed molecular dynamics simulations on the structures of SpCas9, xCas9 and yCas9 to reveal the mechanisms of their PAM recognition and off-target effects. These may provide theoretical guidance for further optimization and modification of CRISPR/Cas9 proteins.
CRISPR-Associated Protein 9/metabolism* ; CRISPR-Cas Systems/genetics* ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; RNA, Guide/genetics* ; Streptococcus pyogenes/metabolism*

Objective: : To explore the clinical efficacy and safety of microvascular decompression (MVD) combined with internal neurolysis (IN) in the treatment of recurrent trigeminal neuralgia (TN) after MVD. Methods: : Sixty-four patients with recurrent TN admitted to the hospital from January 2014 to December 2017 were divided into two groups according to the surgical method. Twenty-nine patients, admitted from January 2014 to December 2015, were treated with MVD alone, whereas 35 admitted from January 2016 to December 2017 were treated with MVD+IN. The postoperative efficacy, complications, and pain recurrence rate of the two groups were analyzed. Results: : The efficacy of the MVD+IN and MVD groups were 88.6% and 86.2%, and the cure rates were 77.1% and 65.5% respectively. There was no statistically significant difference between the two groups (p>0.05). The cure rate (83.3%) of patients in the MVD+IN group, who were only found thickened arachnoid adhesions during the operation that could not be fully released, was significantly higher than that of the MVD group (30.0%) (p<0.05), while the efficacy (91.7% vs. 70%) of the two groups was not statistically different (p>0.05). For patients whose arachnoid adhesions were completely released, there had no significant difference (p>0.05) in the efficacy (87% vs. 94.7%) and recurrence rate (5.0% vs. 11.1%). The incidence of postoperative facial numbness (88.6%) in the MVD+IN group was higher than that in the MVD group (10.3%) (p<0.01). The long-term incidence of facial numbness was not statistically significant (p>0.05). In the 18–36 months follow-up, the recurrence rate of patients in the MVD+IN group (9.7%) and in the MVD group (16%) were not statistically different (p>0.05). Conclusion : A retrospective comparison of patients with recurrent TN showed that both MVD and MVD combined with IN can effectively treat recurrent TN. Compared with MVD alone, MVD combined with IN can effectively improve the pain cure rate of patients with recurrent TN who have only severe arachnoid adhesions. The combination does not increase the incidence of long-term facial numbness and other complications.