Objective:To investigate the ultrasonographic features and genetic etiology of complex talipes equinovarus (TE) in fetuses.Methods:This retrospective study enrolled 95 cases of complex TE (TE complicated by other abnormalities) who were diagnosed by prenatal ultrasound in the Third Affiliated Hospital of Zhengzhou University from March 2018 to December 2022. Chromosome karyotype analysis and/or chromosomal microarray analysis (CMA) [or copy number variation-sequencing (CNV-seq)] were performed on all cases for prenatal genetic diagnosis and those with normal results were further tested by whole exome sequencing (WES). Prenatal ultrasonographic and genetic features of complex TE in fetuses were summarized. Complicated abnormalities in the fetuses were classified into nine categories according to the involved system or site and based on each category these subjects were divided into with or without the corresponding complicated abnormalities groups. Besides, these cases were also divided into single-system and multi-system abnormality groups based on the number of involved systems or sites of complicated abnormalities. The detection rates of WES abnormality (pathogenic or likely pathogenic variants) and the overall detection rate of genetic abnormality [karyotype abnormality detected by chromosome karyotype analysis, pathogenic or likely pathogenic copy number variations (CNVs) detected by CMA (or CNV-seq), and pathogenic or likely pathogenic variation detected by WES] were compared between different groups using Chi-square test or Fisher's exact test. Results:Abnormal chromosome karyotypes were identified in 10 (24.4%) of 41 cases receiving chromosome karyotype analysis, pathogenic and likely pathogenic CNVs were found in seven (7.6%) of 92 cases by CMA (or CNV-seq). WES was performed on 37 cases with negative results of chromosomal karyotype analysis and CMA (or CNV-seq) and the detection rate of pathogenic and likely pathogenic variants was 43.2% (16/37). The detection rate of WES abnormality was higher in the fetuses with musculoskeletal abnormalities than in those without the abnormalities [71.4% (15/21) vs. 1/16, Fisher's exact test, P<0.001], while in those with other postural abnormalities was higher than that in the group without other postural abnormalities [12/16 vs. 19.0% (4/21), Fisher's exact test, P=0.001]. The genetic causes of complex TE were identified in 34.7% (33/95) of the fetuses by the sequential genetic diagnosis using chromosome karyotype analysis, CMA (or CNV-seq), and WES. The overall detection rate of genetic abnormality was higher in the group with multi-system abnormality than in the group with single-system abnormality [48.9% (22/45) vs. 22.0% (11/50), χ2=7.55, P=0.006], in the group with musculoskeletal system abnormalities and without [46.8% (22/47) vs. 22.9% (11/48), χ2=5.98, P=0.014], and in the group with other postural abnormality and without [47.2% (17/36) vs. 27.1% (16/59), χ2=3.99, P=0.046]. Nine cases that were considered isolated TE on initial ultrasound were corrected to a complex diagnosis on subsequent ultrasound examinations. Of all the involved system or site, the neurologic abnormalities were the most diverse (13 kinds) and had a diversity of ultrasound presentations. Conclusions:Genetic diagnosis should be performed when prenatal ultrasound suggests fetal complex TE. WES is conducive to improving the prenatal detection rate of monogenic diseases, especially in fetuses complicated by musculoskeletal abnormalities. Isolated TE fetuses require serial ultrasound examinations to correct the diagnosis in time and genetic testing should be performed if necessary. Additional attention should be paid to the TE fetus for comorbid neurologic abnormalities at the time of ultrasonography to rule out TE as an intrauterine harbinger of neuromuscular disease.

Objective: To investigate the expression of eukaryotic translation initiation factor 2B1(eIF2B1) in HCC and its correlation with clinical prognosis. Methods: Through the follow⁃up of the clinical data of 743 hepatocellular carcinoma patients in the specimen bank ,91 HBV⁃related liver cancer patients with complete follow⁃up information were screened out , and their postoperative tumor tissues were selected. Meanwhile , the adjacent tumor tissues with a distance of more than 2 cm from the tumor margin were collected to make tissue chips. Western blot and immunohistochemical experiments were performed. Image J was used to analyze the proportion of positive cells stained by tissue chip and the gray value of Western blot results. Clinical data and follow⁃up data of included patients were statistically analyzed by R4. 0. 5 software. Results: Immunohistochemical results suggested that eIF2B1 was more strongly expressed in HCC than in para⁃cancerous tissues. Western blot results showed that eIF2B1 was more strongly expressed in HepG2. 2. 15 cells than in HepG2 cells. The expression of eIF2B1 in HCC tissues was correlated with tumor number (P < 0. 05) . Cox multivariate regression analysis showed that the expression of eIF2B1 was an independent risk factor for the overall clinical prognosis of HBV⁃DNA positive patients (HR = 4. 28 ,P = 0. 018) . Conclusion The expression of eIF2B1 is significantly enhanced in HBV⁃associated HCC tissues and is significantly associated with overall survival in HBV DNA positive patients.

Objective:To investigate the effects of poly(A) tails with different lengths on mRNA expression in vitro and the passage stability of transcription template with poly (A) tail in Escherichia coli ( E. coli). Methods:Plasmids with poly(A) tails of 38, 60, 103, 125 and 126 (60 nt+ 6 nt spacer+ 60 nt) nt were designed and constructed. Then the plasmids were linearized by single enzyme digestion and used as transcription template for preparing enhanced green fluorescent protein (EGFP)-mRNA. EGFP-mRNA containing poly(A) tails of different lengths were transfected into 293T cells and the expression of EGFP was detected by flow cytometry. As to stability test, the template plasmids with poly (A) tail of 125 and 126 nt were transformed into E. coli TransStbl3 and Top10 competent cells. Seven clones were selected for culture and plasmid extraction, and then the plasmids were digested by restriction enzyme and detected by capillary electrophoresis. For passage stability, three correctly sequenced clones of each group were selected for continuous passage at 37℃, and the plasmids were extracted and digested every two generations for capillary electrophoresis. At the same time, the correctly sequenced clones of 125 nt group were also passaged at 30℃, and the plasmids were also extracted and digested every two generations for capillary electrophoresis. Results:The transcription templates with poly(A) tail of different lengths were successfully constructed. Flow cytometry showed that the fluorescence expression of the template plasmids with poly (A) tail of 103 and 125 nt were significantly higher than that of 38 and 60 nt. The fluorescence expression of the plasmid with poly (A) tail of 126 nt was significantly higher than that of all other groups. The percentages of stable sequences of the template plasmid with poly(A) tail of 125 nt in TransStbl3 and Top10 competent cells were 76% and 91%, respectively. The results of continuous passage showed that poly(A) tail of 125 nt could be stable to the 4th generation at 37℃ in both TransStbl3 and Top10 competent cells, and stable to the 16th and 10th generations at 30℃. The percentages of stable sequences of the template plasmid with poly(A) tail of 126 nt in TransStbl3 and Top10 competent cells were 95% and 48%, respectively. The results of continuous passage showed that poly(A) tail of 126 nt could be stable to the 12th generation at 37℃ in both TransStbl3 and Top10 competent cells.Conclusions:The length and composition of poly(A) tail in mRNA affected the expression of target protein. Adding a spacer with a length of 6 nt to poly(A) tail and low temperature culture were both helpful to improve the stability of the template plasmid, which provided a reference for the design and preparation of in vitro transcription template of mRNA vaccine.

Objective:To prepare a recombinant hemagglutinin trimer (HA-Tri) vaccine against influenza viruses and to study its immunogenicity in a mouse model.Methods:A stable CHO cell line that could express HA-Tri was constructed. Western blot, single radial immunodiffusion, protein particle size detection and N-glycosylation site analysis were performed for qualitative and quantitative analysis of the recombinant protein. According to the different treatment conditions such as dosage and adjuvant, BALB/c mice were divided into 11 groups and subjected to consistent immunization procedures. Serum neutralizing antibody titers were measured on 56 d after the first immunization to evaluate the immunogenicity of HA-Tri.Results:The constructed CHO cells could secret and express HA-Tri proteins. The HA-Tri proteins were biologically active and capable of forming precipitation rings in the single radial immunodiffusion. The particle size of HA-Tri was approximately 18.79 nm and 10 N-glycosylation sites were detected, including high mannose, complex glycoforms and heterozygous glycoforms. After prime-boost immunization, there was no statistically significant difference in the titers of neutralizing antibodies induced in mice by 3.75 μg of HA-Tri in combination with RFH01 adjuvant and 15 μg of monovalent vaccine stock solution ( P=0.431 2, U=36). Serum antibody titers in the HA-Tri+ RFH01 groups were higher than those in the corresponding HA-Tri groups without RFH01 adjuvant, and the highest titer was induced in the 15 μg HA-Tri+ RFH01 group, which was 1 280. Conclusions:The recombinant HA-Tri protein was successfully prepared. HA-Tri in combination with RFH01 adjuvant could induce humoral immune responses against influenza viruses in BALB/c mice, which would provide reference for the development of influenza virus recombinant subunit vaccines.

Objective:To evaluate the immunogenicity of a quadrivalent subunit vaccine combined with RFH01 adjuvant in a mouse model.Methods:Identification tests were performed on four monovalent influenza virus subunit vaccine stock solutions according to the methods described in Part 3 of the Chinese Pharmacopoeia 2020 Edition. In the study of the quadrivalent subunit vaccine combined with RFH01 adjuvant, 460 female BALB/c mice (6-8 weeks old) were randomly divided into 46 groups including experimental groups, vaccine control group, negative control group and blank group with 10 mice in each group. In the study of the quadrivalent subunit vaccine in old and young mice, 80 female 10-month-old and 80 female 10-week-old BALB/c mice were randomly divided into 16 groups ( n=10) including monovalent influenza virus vaccine group, quadrivalent subunit vaccine group, quadrivalent subunit vaccine+ RFH01 adjuvant group, chicken embryo quadrivalent split vaccine control group and PBS group. All mice were immunized by intramuscular injection. At 21 d after the primary immunization, a booster immunization was conducted using the same strategy. Blood samples were collected at 21 d and 42 d after the primary immunization for serum separation. Haemagglutination inhibition (HI) test was performed to detect the antibody levels in mouse serum samples. Results:After the booster immunization, the positive conversion rates in all vaccine+ RFH01 adjuvant groups reached 100%, and the geometric mean titers (GMTs) of serum antibodies were significantly higher than those of the vaccine groups without RFH01 adjuvant. There were significant differences in serum antibody titers between the monovalent/quadrivalent subunit vaccine groups with and without RFH01 adjuvant. After the booster immunization, the titers of serum antibodies against H1N1, H3N2, B/Victoria and B/Yamagata in the 10-week-old mice were significantly higher than those in the 10-month-old mice.Conclusions:The monovalent and quadrivalent influenza virus vaccines in combination with RFH01 adjuvant could elicit higher antibody titers in young (6-10 weeks old) and old (10 months old) mice, showing good immunogenicity.

Objective:To express the head domain of influenza A virus hemagglutinin (HA) in a prokaryotic expression system and to evaluate its immunogenicity.Methods:The genes encoding the HA head domains of H1N1 and H3N2 influenza viruses were cloned into pET-22b(+ ) prokaryotic expression plasmid. After the induction with IPTG, the fusion proteins rH1N1-HA and rH3N2-HA containing HA head domain and His-tag were expressed and obtained from E. coli BL21. SDS-PAGE and Western blot was used to verify the expression of the recombinant proteins. Rabbits were immunized with multiple doses of the purified recombinant proteins to obtain polyclonal antibodies against the HA head domains of H1N1 and H3N2. The immunogenicity of the recombinant proteins was evaluated in BALB/c mice. Results:rH1N1-HA and rH3N2-HA induced protective antibodies (geometric mean titer ≥40) in mice and could be used as protective antigens. Polyclonal antibodies against rH1N1-HA and rH3N2-HA could be used as important materials for Western blot, ELISA and other immunological assays.Conclusions:The HA head domains prepared in this study could be used as protective antigens to induce protective antibodies in mice. Polyclonal antibodies against the HA head domains could be used for immunological and serological studies of influenza A viruses.

BACKGROUND: Zirconia inlays have good mechanical, biocompatible and aesthetic properties in the field of dental prosthodontics, but there is no consensus on the standards of zirconia inlays for clinical cavity design, dental preparation and selection of bonding materials. OBJECTIVE: To investigate the stress distribution and characteristics of the bonding interface, tooth and periodontal tissues of the zirconia inlay model after 3 M RelyX Unicem or vario-link resin adhesive bonding. METHODS: Micro-CT was used to scan the right mandibular third molars. A three-dimensional finite element model of MOD zirconia inlay with different adhesives (3 M RelyX Unicem resin binder, vario-link resin binder) and cavity types (2, 3, 4 mm in the depth of the joint cavity) was constructed by software Mimics, Goemagic Studio and NX 10.0. Using ANSYS Workbench mesh generation, the stress distribution of each model was analyzed after loading 10 N·mm torque, 45° 175 N, 90° 600 N. RESULTS AND CONCLUSION: (1) After loading 10 N·mm torque, the bonding agent equivalent stress and root surface equivalent stress of the model with 3 mm cavity depth were largest, so the stress on the bonding interface, crown and root was largest. (2) After loading 175 N at 45° lingual direction, the bonding interface of the model with 4 mm cavity depth undertook a higher stress. The stress values of the root and periodontal tissue of 3 M RelyX Unicem resin bonding agent model with the same cavity depth were higher. (3) After loading 600 N at 90°, the root force of the model with 3 mm cavity depth was largest. The crown, root and periodontal tissue of the 3 M RelyX Unicem resin bonding agent model with the same cavity depth undertook a higher stress. The bonding interface of the vario-link resin bonding agent model was under greater stress. (4) The regions of stress concentration areas included the bifurcation zone of the root, the inlay edge line, the roof of pulp chamber, the gingival wall, 1/3 mesial part of the buccal surface near the neck, and 1/3 mesial part of the lingual surface near the neck. (5) All these findings indicate that vario-link resin adhesive and 3 M RelyX Unicem resin adhesive are suitable for the bonding of zirconia inlay, but the vario-link adhesive strength is larger and the bonding interface stress is larger. The factors such as cavity design, residual tooth tissue resistance, retention shape, and periodontal support should be considered comprehensively. The optimization of cavity design, tooth preparation, cushion bottom and high inlay should be adopted, in order to improve the long-term repair effect of zirconia inlay. However, further clinical trials are needed to verify the results of three-dimensional finite element model.

Objective To analyze the expression of interleukin 16 (IL-16) in atherosclerosis (AS) patients, and to study the roles of IL-16in the pathogenesis of AS.Methods Thirty AS patients in Affiliated Hospital of Jining Medical College from August 2015to August 2016were randomly selected as the case group and twenty-nine healthy subjects were selected as the healthy control group.Peripheral blood of the subjects were collected.IL-16levels were determined with enzyme-linked immunosorbent assay (ELISA).Reverse transcriptase-polymerase chain reaction was applied to analyze IL-16mRNA level.IL-16expression in the atherosclerotic plaque samples was detected with immunohistochemical analysis.IL-16expression in aortic atherosclerotic plaque of AS patients and atherosclerotic ApoE-/-mice were analyzed by immunohistochemical staining.The aortic plaque changes of AS mice injected intraperitoneally with recombinant IL-16were detected.Results Both IL-16protein levels and IL-16mRNA levels were higher in case group than those of healthy control group, the difference was statistically significant (P＜0.05).The IL-16mRNA was highly expressed in the atherosclerotic plaque.The aortic plaque area of the mice underwent IL-16intraperitoneal injection were decreased while the plaque stability increased.Conclusion IL-16levels elevated in both AS patients and AS mice, which suggested that IL-16might play aprotective role against AS.

Objective To observe the effect of comprehensive rehabilitation nursing intervention in patients with hypertensive intracerebral hemorrhage complicated with hemiplegia and its effect on neurological function.Methods A total of 120 hypertensive intracerebral hemorrhage complicated with hemiplegia were selected as research objects,and were divided into control group and observation group according to random number table method,with 60 cases in each group.The patients in the control group were treated with routine rehabilitation,and the patients in the observation group were given comprehensive rehabilitation nursing on the basis of routine rehabilitation.The levels of muscle strength,neurological function and recovery efficacy were evaluated before and 6 months after nursing.Results There was no significant difference in muscle strength between the two groups before treatment (P ＞ 0.05).After the implementation,muscle strength of two groups were increased,and the observation group was higher than the control group (P ＜ 0.05).The levels of S100B and MBP were lower,and the level of BDNF was higher than nursing before and that in the control group (P ＜0.05).The score of NIHSS was lower and the score of Barthel index was higher than nursing before and that in the control group (P ＜0.05).Conclusion Comprehensive rehabilitation care intervention can effectively enhance the patient's muscle strength level,improve limb function,reduce neurological damage,improve self-care ability,and reduce the impact of sequelae for patients with hypertensive intracerebral hemorrhage complicated with hemiplegia.

Objective To observe the effect of comprehensive rehabilitation nursing intervention in patients with hypertensive intracerebral hemorrhage complicated with hemiplegia and its effect on neurological function.Methods A total of 120 hypertensive intracerebral hemorrhage complicated with hemiplegia were selected as research objects,and were divided into control group and observation group according to random number table method,with 60 cases in each group.The patients in the control group were treated with routine rehabilitation,and the patients in the observation group were given comprehensive rehabilitation nursing on the basis of routine rehabilitation.The levels of muscle strength,neurological function and recovery efficacy were evaluated before and 6 months after nursing.Results There was no significant difference in muscle strength between the two groups before treatment (P ＞ 0.05).After the implementation,muscle strength of two groups were increased,and the observation group was higher than the control group (P ＜ 0.05).The levels of S100B and MBP were lower,and the level of BDNF was higher than nursing before and that in the control group (P ＜0.05).The score of NIHSS was lower and the score of Barthel index was higher than nursing before and that in the control group (P ＜0.05).Conclusion Comprehensive rehabilitation care intervention can effectively enhance the patient's muscle strength level,improve limb function,reduce neurological damage,improve self-care ability,and reduce the impact of sequelae for patients with hypertensive intracerebral hemorrhage complicated with hemiplegia.