Objective:To develop a camelid single-domain antibody (SdAb) recognizing linear B-cell epitopes in glutamate dehydrogenase of Clostridium difficile(CD-GDH), and to apply it in Western blot and ELISA. Methods:Purified recombinant CD-GDH was used as bait to screen phage-displayed camelid SdAb library and obtain positive clones. Then those clones were confirmed by Western blot, and their variable domain of heavy chain of heavy chain antibody(VHH) nucleotide sequence were determined. The VHH sequence was synthesized after codon optimization and cloned into the expression vector pET28a. The SdAb was then expressed and purified, and its ability to detect CD-GDH protein in multiple assays was further explored.Results:Six positive clones were obtained, among which clone GA4 was chosen for recombinant expression in Escherichia coli and further purification. The purified GA4 binded well with CD-GDH with a Kd value of 3 nmol/L. In Western blot and ELISA, GA4 was proven to be able to selectively detect both recombinant and endogenous CD-GDH. Conclusions:A camelid SdAb targeting a linear B-cell epitope in CD-GDH is successfully developed, which provides a very useful tool for detecting CD-GDH.

Objective:To investigate the effect of ribonucleotide reductase regulatory subunit M2 (RRM2) on the malignant biological behavior and aerobic glycolysis of gastric cancer cells by regulating cyclin-dependent kinase (CDK) 1.Methods:Human gastric cancer MKN-45 cells were divided into si-NC group (transfected with blank fragment) , CoCl 2+si-NC group (hypoxia control transfected with blank fragment) , CoCl 2+si-RRM2 group (hypoxia with RRM2 silencing) , CoCl 2+si-RRM2+pcDNA3.1 NC group (hypoxia with RRM2 silencing and blank vector) and CoCl 2+si-RRM2+pcDNA3.1 CDK1 group (hypoxia with RRM2 silencing and CDK1 overexpression) . The mRNA relative expression levels of RRM2 and CDK1 were analyzed by real time fluorescent quantitative reverse transcription PCR. Co-immunoprecipitation (CoIP) was used to analyze the interaction between RRM2 and CDK1 protein. MTT assay was used to analyze the proliferation activity of cells. The cell migration distance was detected by cell scratch assay. Cell apoptosis was detected by flow cytometry. Adenosine triphosphate (ATP) and glucose kit were used to detect ATP production and glucose consumption. The protein expressions of ENO1, RRM2, HK2, PKM2, GLUT1 and p-CDK1/CDK1 were detected by Western blotting. Results:Real time fluorescent quantitative reverse transcription PCR results showed that the relative expression levels of CDK1 mRNA in si-NC group, CoCl 2+si-NC group and CoCl 2+si-RRM2 group were 1.01±0.15, 1.30±0.06 and 0.51±0.18, and the relative expression levels of RRM2 mRNA were 1.03±0.32, 1.59±0.28 and 0.44±0.17, respectively, and there were statistically significant differences ( F=25.52, P=0.001; F=14.47, P=0.005) . The mRNA expressions of RRM2 and CDK1 in CoCl 2+si-NC group were higher than those in si-NC group. Compared with the si-NC group and the CoCl 2+si-NC group, the mRNA expressions of RRM2 and CDK1 were lower in the CoCl 2+si-RRM2 group (all P<0.05) . CoIP results showed that there was interaction between RRM2 and CDK1. MTT assay, cell scratch assay and flow cytometry showed that the cell proliferation activity of si-NC group, CoCl 2+si-NC group, CoCl 2+si-RRM2 group, CoCl 2+si-RRM2+pcDNA3.1 NC group and CoCl 2+si-RRM2+pcDNA3.1 CDK1 group were 1.04±0.01, 1.18±0.04, 0.84±0.03, 0.81±0.03 and 0.93±0.05, respectively. The cell migration distances were (301.83±2.75) , (369.67±0.76) , (176.50±6.38) , (175.83±3.69) , (254.17±1.61) μm, respectively. The apoptosis rates were 8.05%±0.21%, 5.75%± 0.20%, 28.28%±0.04%, 30.18%±1.51% and 17.79%±0.22%, respectively, all with statistically significant differences ( F=73.82, P<0.001; F=1 600.01, P<0.001; F=787.15, P<0.001) . Compared with the si-NC group and CoCl 2+si-NC group, the proliferation and migration ability of cells in the CoCl 2+si-RRM2 group, CoCl 2+si-RRM2+pcDNA3.1 NC group and CoCl 2+si-RRM2+pcDNA3.1 CDK1 group were weaker, and the apoptosis rates were higher (all P<0.05) . Compared with the CoCl 2+si-RRM2+pcDNA3.1 NC group, the proliferation and migration ability of cells in the CoCl 2+si-RRM2+pcDNA3.1 CDK1 group were stronger, and the apoptosis rate was lower (all P<0.05) . The results of ATP and glucose detection showed that there were statistically significant differences in the amount of ATP production and glucose consumption among the above five groups ( F=12.53, P<0.001; F=19.21, P<0.001) . Compared with the si-NC group, the glucose consumption of cells was lower in the CoCl 2+si-RRM2+pcDNA3.1 CDK1 group ( P<0.05) . Compared with the CoCl 2+si-NC group, the ATP production and glucose consumption of cells in the CoCl 2+si-RRM2+pcDNA3.1 CDK1 group were lower (both P<0.05) . Compared with the CoCl 2+si-RRM2+pcDNA3.1 NC group, the ATP production and glucose consumption of the CoCl 2+si-RRM2+pcDNA3.1 CDK1 group were higher (both P<0.05) . Western blotting showed that there were statistically significant differences in the protein expressions of ENO1, RRM2, HK2, PKM2, GLUT1, and p-CDK1/CDK1 among the above five groups (all P<0.001) . Compared with the si-NC group and the CoCl 2+si-NC group, the protein expressions of ENO1, RRM2, HK2, PKM2, GLUT1 and p-CDK1/CDK1 in the CoCl 2+si-RRM2+pcDNA3.1 CDK1 group were lower (all P<0.05) . Compared with the CoCl 2+si-RRM2+pcDNA3.1 NC group, the protein expressions of ENO1, RRM2, PKM2, GLUT1 and p-CDK1/CDK1 in the CoCl 2+si-RRM2+pcDNA3.1 CDK1 group were higher (all P<0.05) . Conclusions:Silencing RRM2 can inhibit the malignant biological behavior of gastric cancer cells and the occurrence of aerobic glycolysis by regulating CDK1.

Objective To explore the influence of heparin sodium on early recovery after lumbar surgery and the risk factors of deep vein thrombosis(DVT)of lower extremities,and build a prediction model for DVT after lumbar surgery based on the risk factors.Methods A total of 276 patients who underwent lumbar surgery and were treated with heparin sodium in The First Affiliated Hospital of Naval Medical University from January 2020 to January 2023 were selected as research objects.Activated partial thromboplastin time(APTT),prothrombin time(PT)and functional indexes of lumbar spine were recorded before and after treatment.DVT of lower extremities was detected by ultrasound during postoperative hospitalization,and then the patients were divided into DVT group and non-DVT group.The clinical data and laboratory indicators of all patients were collected.The risk factors of DVT after lumbar surgery were analyzed by logistic regression analysis.A postoperative DVT risk prediction model based on Logistic regression analysis was constructed and validated.Results APTT and PT after operation were higher than those before operation(P<0.05).The score of Oswestry Disability Index(ODI)3 months after surgery was lower than that before surgery(t=30.661,P<0.05).The incidence of DVT was 14.86%(41/276).Blood transfusion,bed rest≥5 d,proportion of general anesthesia,intraoperative blood loss,body mass index(BMI)and D-dimer level in the DVT group were higher than those in the non-DVT group(P<0.05).Logistic regression analysis showed that intraoperative blood loss,BMI,abnormal D-dimer level,intraoperative blood transfusion,and bed rest≥5 d were all risk factors for DVT after lumbar surgery(P<0.05).logit(P)=9.762+1.425×intraoperative blood loss+1.212×BMI+0.856×intraoperative blood transfusion+1.105×bed rest+1.671×D-dimer.Hosmer-Lemeshow test showed that the model had a high goodness of fit(χ2=4.025,P=0.473).The sensitivity,specificity and area under curve(AUC)of the constructed prediction model for postoperative DVT were 91.80%,70.40%and 0.836(95%CI:0.698-0.948),respectively.Conclusion Heparin sodium can improve blood circulation and lumbar function and prolong clotting time in patients with lumbar surgery.Intraoperative blood loss,BMI,abnormal D-dimer,intraoperative blood transfusion,and bed rest≥5 d are all risk factors for the occurrence of DVT after lumbar surgery.The prediction model based on the above risk factors has high predictive value for the occurrence of DVT after lumbar surgery.

Objective:To investigate the effects of ascites grading and the application of non-selective beta-blockers (NSBBs) on the 1-year prognosis of acute-on-chronic liver failure (ACLF).Methods:1 386 ascitic cases with ACLF were graded and followed up for one year. The 1-year prognostic effect of ascites grade and NSBBs was analyzed on ACLF by the Kaplan Meier Log-rank test, Cox stepwise regression, and multivariate regression.The t-test, Mann-Whitney U, or Kruskal-Wallis test were used for intergroup comparison of measurement data. The χ2 test was used for intergroup comparison of numerical data. Results:The incidence rate of ascites at admission was 77.56% in 1 386 ACLF cases. The Log-rank (Mantel-Cox) of the 1-year survival curve test for 1 386 ACLF patients with ascites grade was 21.384, P<0.01. Multivariate regression and Cox stepwise regression analysis showed that ascites grade, age, gastrointestinal bleeding, pulmonary infection, acute kidney injury, prothrombin activity (PTA), urea, MELD-Na score, and the use of NSBBs were closely related to the 1-year prognosis of ACLF. The log rank (Mantel-Cox) of NSBBs treatment in the grade 2/3 ascites group was 6.113, P=0.013, and the difference was statistically significant, suggesting that NSBBs treatment can help improve the 1-year survival rate in ACLF patients with grade 2 and 3 ascites. Conclusions:Ascites grading and the use of NSBBs affect the prognostic factor of ACLF at one year. NSBBs may be beneficial for the long-term prognosis of ACLF, and treatment can be continued in patients who have already received NSBBs prior to the onset of ACLF.

Objective:To study the lipid metabolism and its relationship with iodine nutrition status in patients with papillary thyroid carcinoma (PTC) and benign thyroid nodules.Methods:A case-control study was conducted on patients with thyroid nodules initially diagnosed at Harbin Medical University Cancer Hospital from November 2015 to April 2019. Basic information, thyroid function indicators [thyroid stimulating hormone (TSH), free triiodothyronine (FT 3), free thyroxine (FT 4), thyroglobulin antibodies (TgAb), thyroid peroxidase antibodies (TPOAb)], and pathological diagnosis results were collected from all subjects. Fasting venous blood and morning urine samples were collected for serum iodine concentration (SIC), urine iodine concentration (UIC) and blood lipid indicators [total cholesterol (TC), triglyceride (TG), low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C), lipoprotein (a), apolipoprotein A1 (ApoA1), apolipoprotein B (ApoB), apolipoprotein E (ApoE), and non-esterified fatty acid (NEFA)] measurements. Results:A total of 1 090 subjects were included, including 907 PTC patients and 183 benign thyroid nodule patients. The UIC [ M ( Q1, Q3)] for the PTC group and benign thyroid nodule group were 143.36 (94.08, 227.94) and 146.28 (112.89, 236.07) μg/L, respectively, with statistically significant differences between the groups ( Z = 4.16, P = 0.042). Among PTC patients with different clinical pathological features, those with lymph node metastasis had higher FT 3 levels than those without lymph node metastasis ( t = 5.42, P = 0.021). The levels of TSH, TgAb, and TPOAb in patients with PTC combined with autoimmune thyroid disease (AITD) were higher than those without AITD ( Z = 11.87, 81.55, 475.96, P < 0.05). There was no statistically significant difference in the comparison of various blood lipid indicators ( P > 0.05). The correlation analysis showed that SIC in patients with thyroid nodules was positively correlated with FT 3, FT 4, ApoB, lipoprotein (a), NEFA, TG, LDL-C, and LDL-C/HDL-C ( P < 0.05). FT 3 was positively correlated with NEFA and LDL-C/HDL-C ( r = 0.12, 0.09, P < 0.05), and negatively correlated with ApoA1, TC, and HDL-C ( r = - 0.14, - 0.14, - 0.15, P < 0.001). FT 4 was positively correlated with NEFA ( r = 0.11, P < 0.001), and negatively correlated with TG ( r = - 0.10, P = 0.003). According to the iodine nutritional level, the ApoE level of PTC patients in the SIC < 45 μg/L group was higher than that in the 45 - 90 μg/L group ( P < 0.05). The levels of LDL-C and ApoB in patients with benign thyroid nodules in the SIC > 90 μg/L group were higher than those in the 45 - 90 μg/L group ( P < 0.05). The ApoE level of patients with benign thyroid nodules in the UIC < 100 μg/L group was significantly higher than that in the 100 - 299 and ≥300 μg/L groups ( P < 0.05). There was no statistically significant difference in thyroid function indicators of patients with PTC and benign thyroid nodules between different levels of SIC and UIC ( P > 0.05). Conclusions:Compared to benign thyroid nodules, PTC does not significantly affect patients' blood lipid levels. In patients with PTC and benign thyroid nodules, insufficient or excessive iodine nutrition can affect lipid metabolism.

Objective:To study the lipid metabolism and its relationship with iodine nutrition status in patients with papillary thyroid carcinoma (PTC) and benign thyroid nodules.Methods:A case-control study was conducted on patients with thyroid nodules initially diagnosed at Harbin Medical University Cancer Hospital from November 2015 to April 2019. Basic information, thyroid function indicators [thyroid stimulating hormone (TSH), free triiodothyronine (FT 3), free thyroxine (FT 4), thyroglobulin antibodies (TgAb), thyroid peroxidase antibodies (TPOAb)], and pathological diagnosis results were collected from all subjects. Fasting venous blood and morning urine samples were collected for serum iodine concentration (SIC), urine iodine concentration (UIC) and blood lipid indicators [total cholesterol (TC), triglyceride (TG), low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C), lipoprotein (a), apolipoprotein A1 (ApoA1), apolipoprotein B (ApoB), apolipoprotein E (ApoE), and non-esterified fatty acid (NEFA)] measurements. Results:A total of 1 090 subjects were included, including 907 PTC patients and 183 benign thyroid nodule patients. The UIC [ M ( Q1, Q3)] for the PTC group and benign thyroid nodule group were 143.36 (94.08, 227.94) and 146.28 (112.89, 236.07) μg/L, respectively, with statistically significant differences between the groups ( Z = 4.16, P = 0.042). Among PTC patients with different clinical pathological features, those with lymph node metastasis had higher FT 3 levels than those without lymph node metastasis ( t = 5.42, P = 0.021). The levels of TSH, TgAb, and TPOAb in patients with PTC combined with autoimmune thyroid disease (AITD) were higher than those without AITD ( Z = 11.87, 81.55, 475.96, P < 0.05). There was no statistically significant difference in the comparison of various blood lipid indicators ( P > 0.05). The correlation analysis showed that SIC in patients with thyroid nodules was positively correlated with FT 3, FT 4, ApoB, lipoprotein (a), NEFA, TG, LDL-C, and LDL-C/HDL-C ( P < 0.05). FT 3 was positively correlated with NEFA and LDL-C/HDL-C ( r = 0.12, 0.09, P < 0.05), and negatively correlated with ApoA1, TC, and HDL-C ( r = - 0.14, - 0.14, - 0.15, P < 0.001). FT 4 was positively correlated with NEFA ( r = 0.11, P < 0.001), and negatively correlated with TG ( r = - 0.10, P = 0.003). According to the iodine nutritional level, the ApoE level of PTC patients in the SIC < 45 μg/L group was higher than that in the 45 - 90 μg/L group ( P < 0.05). The levels of LDL-C and ApoB in patients with benign thyroid nodules in the SIC > 90 μg/L group were higher than those in the 45 - 90 μg/L group ( P < 0.05). The ApoE level of patients with benign thyroid nodules in the UIC < 100 μg/L group was significantly higher than that in the 100 - 299 and ≥300 μg/L groups ( P < 0.05). There was no statistically significant difference in thyroid function indicators of patients with PTC and benign thyroid nodules between different levels of SIC and UIC ( P > 0.05). Conclusions:Compared to benign thyroid nodules, PTC does not significantly affect patients' blood lipid levels. In patients with PTC and benign thyroid nodules, insufficient or excessive iodine nutrition can affect lipid metabolism.

Objective:To develop a camelid single-domain antibody (SdAb) recognizing linear B-cell epitopes in glutamate dehydrogenase of Clostridium difficile(CD-GDH), and to apply it in Western blot and ELISA. Methods:Purified recombinant CD-GDH was used as bait to screen phage-displayed camelid SdAb library and obtain positive clones. Then those clones were confirmed by Western blot, and their variable domain of heavy chain of heavy chain antibody(VHH) nucleotide sequence were determined. The VHH sequence was synthesized after codon optimization and cloned into the expression vector pET28a. The SdAb was then expressed and purified, and its ability to detect CD-GDH protein in multiple assays was further explored.Results:Six positive clones were obtained, among which clone GA4 was chosen for recombinant expression in Escherichia coli and further purification. The purified GA4 binded well with CD-GDH with a Kd value of 3 nmol/L. In Western blot and ELISA, GA4 was proven to be able to selectively detect both recombinant and endogenous CD-GDH. Conclusions:A camelid SdAb targeting a linear B-cell epitope in CD-GDH is successfully developed, which provides a very useful tool for detecting CD-GDH.

Objective:To investigate the effects of ascites grading and the application of non-selective beta-blockers (NSBBs) on the 1-year prognosis of acute-on-chronic liver failure (ACLF).Methods:1 386 ascitic cases with ACLF were graded and followed up for one year. The 1-year prognostic effect of ascites grade and NSBBs was analyzed on ACLF by the Kaplan Meier Log-rank test, Cox stepwise regression, and multivariate regression.The t-test, Mann-Whitney U, or Kruskal-Wallis test were used for intergroup comparison of measurement data. The χ2 test was used for intergroup comparison of numerical data. Results:The incidence rate of ascites at admission was 77.56% in 1 386 ACLF cases. The Log-rank (Mantel-Cox) of the 1-year survival curve test for 1 386 ACLF patients with ascites grade was 21.384, P<0.01. Multivariate regression and Cox stepwise regression analysis showed that ascites grade, age, gastrointestinal bleeding, pulmonary infection, acute kidney injury, prothrombin activity (PTA), urea, MELD-Na score, and the use of NSBBs were closely related to the 1-year prognosis of ACLF. The log rank (Mantel-Cox) of NSBBs treatment in the grade 2/3 ascites group was 6.113, P=0.013, and the difference was statistically significant, suggesting that NSBBs treatment can help improve the 1-year survival rate in ACLF patients with grade 2 and 3 ascites. Conclusions:Ascites grading and the use of NSBBs affect the prognostic factor of ACLF at one year. NSBBs may be beneficial for the long-term prognosis of ACLF, and treatment can be continued in patients who have already received NSBBs prior to the onset of ACLF.

Objective To statistically analyze the allergen-specific IgE test results for allergic patients and observe the distribution of allergens and their prevalence trends.Methods Allergen-specific IgE test results were collected from 12 486 patients with suspected allergic diseases who attended the outpatient clinic of the Department of Dermatology of the Second Affiliated Hospital of Air Force Medical University from January 2018 to December 2023,and the distribution of the positivity rate of various allergens and their differences among different age,gender and seasonal subgroups were statistically analyzed.Results Allergen-specific IgE positive results were found in 5 109 cases out of 12 486 patients with suspected allergic diseases,with a positive rate of 40.92%.Between different gender groups,the allergen positivity rate were 45.41%(2 371/5 221)in males and 37.69%(2 738/7 265)in females,respectively,and the difference in positivity rate between males and females was statistically significant(x2=74.99,P<0.001).The allergen positivity rate among different age groups was highest in the 3～<6 years group(60.00%),followed by the 6～<18 years group(54.92%),and lowest in the≥60 years group(29.18%),with statistically significant differences in allergen positivity rates among different age groups(x2=344.97,P<0.001).The top three overall allergen positivity rates were milk(11.73%),dwarf ragweed/artemisia/rudolfia/quinoa(11.43%)and house dust mite(10.82%),and the positivity rates of milk and house dust mite had a tendency to increase year by year.The highest allergen positivity rate among groups in different seasons was in summer(42.28%),followed by fall(41.32%)and lowest in winter(38.31%),and the difference in allergen positivity rates among the four seasons was statistically significant(x2=9.09,P=0.028).Dwarf ragweed/artemisia/rutabaga/quinoa had the highest positivity rate in the fall(15.75%),while milk(14.59%)and egg white(2.58%)had the highest positivity rates in the summer,and the differences in the positivity rates of dwarf ragweed/artemisia/rutabaga/quinoa,milk and egg white between the four seasons were statistically significant(x2=92.50,70.45,8.10,all P<0.05).Multiple positives were present for allergen-specific IgE,with 22.18%single positives and 18.92%double and more than double positives.Positive test results for ingestion were mainly distributed in the lower levels,while positive test results for inhalation were more distributed in the higher levels,especially for dwarf ragweed/artemisia/rutabaga/quinoa,which were most distributed in level 6.Conclusion Different types of allergens have different distribution characteristics among different genders,ages and seasons,the distribution levels of positive results are different,and there are multiple positive results.Based on the distribution characteristics of allergens in the region,rational arrangement of the dietary and living shoule be mode to avoid the risks and to reduce the probability of the occurrence of allergic diseases.

Objective:To analyze the effects of exposure to life events and social support levels during different stages of pregnancy on low birth weight in offspring.Methods:From 2021 to 2023, pregnant women in early pregnancy who were registered with health cards in Linping District, Hangzhou City, were recruited and followed up. The Life Events Scale for Pregnant Women (LESPW) and Social Support Rating Scale (SSRS) were used to evaluate the exposure to life events and social support levels of pregnant women in early, middle and late pregnancy, respectively. A generalized linear model was used to analyze the impact and critical period of exposure to prenatal life events and social support levels on the risk of low birth weight in offspring.Results:A total of 3 663 pregnant women with ages M ( Q1, Q3) of 29.18 (26.59, 32.21) years were included in this study. 116 cases (3.17%) of infants with low birth weight were delivered. Generalized linear model analysis showed no correlation between the level of social support in early, middle and late pregnancy and low birth weight in offspring (all P>0.05). The risk of low birth weight in offspring of pregnant women with high exposure to early pregnancy life events was 1.78 times higher than that of the low exposure group ( RR=1.78, 95% CI: 1.13-2.73, P=0.010), and there was no association in the middle and late stages of pregnancy (both P>0.05). Pregnant women with high exposure to early life events and low levels of social support had a 2.08-fold higher risk of low birth weight in their offspring compared to the low exposure group ( RR=2.08, 95% CI: 1.17-6.78, P=0.010), while this situation was not associated with high social support stratification. Conclusion:Exposure to life events during pregnancy has an early window effect on birth weight. Increased exposure to life events during early pregnancy is associated with a higher risk of low birth weight in offspring. This association is more pronounced in pregnant women with low social support.