To prepare a recombinant adeno-associated virus(rAAV9-ZsGreen1-EsASP)capable of expressing the aspartic protease protein of Eimeria stiedai(E.stiedai),and explore its in vitro ac-tivity.The EsASP gene was amplified by PCR,and recombinant adeno-associated viral vector pAAV-IRES-ZsGreen1 was combined with the EsASP gene using homologous recombination to construct the pAAV-ZsGreen1-EsASP expression plasmid;pAAV-ZsGreen1-EsASP expression plasmid,pHelper,and pAAV-RC9 plasmids were cotransfected into HEK-293T cells by liposomal transfection to package and produce recombinant adeno-associated virus rAAV9-ZsGreen1-EsASP capable of expressing EsASP protein.rAAV9-ZsGreen1-EsASP was purified using chloroform treatment-PEG/NaCl precipitation-chloroform extraction method,the purity of the virus was iden-tified by silver staining,the virus morphology was observed by TEM,and virus titer was detected by qRT-PCR;the purified recombinant virus was further infected into HEK-293T cells,and EsASP expression was detected by observing green fluorescent protein ZsGreen1 and Western blot method.The results indicated that double enzyme digestion and DNA sequencing confirmed that the EsASP gene had been successfully constructed into the pAAV-IRES-ZsGreen1 expression plas-mid.The expression of green fluorescent protein in HEK-293T cells suggested that co-transfection was successful.Western blot results of cell protein preparation showed that EsASP protein was successfully expressed;The purified recombinant viral capsid proteins show three distinct bands(VP1-3).The purified rAAV9-ZsGreen1-EsASP was uniform in size,around 20 nm,and had a titer higher than 1.0 × 1012 vg/mL;Green fluorescent protein expression was observed after infection of HEK-293T cells with the recombinant virus,and EsASP expression was detected by Western blot.The results suggest that rAAV9-ZsGreen1-EsASP with in vitro infectious activity was suc-cessfully obtained,providing a material basis for the development of a novel vaccine against Eimer-ia stiedai.

To understand the infection status of main intestinal protozoa in BALB/c mice and pro-vide a basis for further control of intestinal protozoa infection.Five hundred and forty BALB/c mice provided by four domestic suppliers of BALB/c mice were detected for intestinal protozoa,in which 140 from supplier A,130 from supplier B,135 from supplier C,and 135 from supplier D,re-spectively.Fresh faecal samples were collected from each mouse separately to extract the genome and amplified by nested PCR based on primers for the 18S rRNA gene sequences of Pent-atrichomonas hominis(P.hominis)and Cryptosporidium tyzzeri(C.tyzzeri),and the 16S-like rRNA gene sequence of Tritrichomonas muris(T.muris)and sequenced.The results showed that the total intestinal protozoan infection rate was 7.1％(10/140)in 140 mice faecal samples provided by supplier A.Among them,the positivity rate of T.muris was 7.1％(10/140),C.tyzzeri was 2.1％(3/140),and P.hominis was 7.1％(10/140),the co-infection rate of two intestinal protozoa was 7.1％(10 mice:T.muris+P.hominis),and three intestinal protozoa was 2.1％(3 mice:T.muris+P.hominis+C.tyzzeri).The total intestinal protozoan infection rate in 135 mice faecal samples provided by supplier C was 7.4％,in which,7.4％(10/135)was positive for T.muris.There are no intestinal protozoa to be detected in 130 mice faecal samples from supplier B and 135 mice faecal samples from supplier D.The homology analysis showed that the homology of ampli-fied sequence of T.muris,P.hominis and C.tyzzeri was 98.52％,98.27％and 99.87％compared with published sequence of GenBank No:AY886846.1,GenBank No:AF156964.1 and GenBank No:KJ000486.1,which was clustered as an independent branch by phylogenetic analysis respec-tively.In conclusion,there are intestinal protozoan infection in BALB/c mice in some animal sup-pliers.The co-infections of more than 3 parasites such as T.muris,P.hominis and C.tyzzeri has been found.It will provide a basis for control of intestinal protozoa infection in BALB/c mice in the future.

Objective:To construct a risk prediction model for radiation dermatitis in the perineum of cervical cancer patients undergoing radiotherapy, and verify the predictive effect of the model, so as to provide theoretical basis for reducing the risk of perineal radiation dermatitis in clinical practice.Methods:A prospective study design was used to conveniently select 247 cervical cancer patients who received radiotherapy at Tianjin Medical University Cancer Institute & Hospital from January 2022 to March 2023 as the modeling group. They were divided into a group with perineal radiodermatitis ( n = 130) and a group without perineal radiodermatitis ( n = 117). The data of the two groups were compared, and a risk prediction model was constructed based on the results of logistic regression analysis. Used the area under the receiver operating characteristic (ROC) curve to test the predictive performance of the model. From May to November 2023, 109 patients were selected as the validation group to conduct external validation of the model. Results:Among the 247 patients in the modeling group, the age of the group with perineal radiodermatitis was (45.31 ± 7.25) years old, and the age of the group without perineal radiodermatitis was (46.70 ± 6.43) years old. The incidence of perineal radiation dermatitis during radiotherapy in the modeling group was 52.6% (130/247), while in the validation group it was 44.0% (48/109). The final predictive variables included in the model were high blood sugar or not ( OR = 0.304, 95% CI 0.174-0.530), vaginal bleeding/discharge or not ( OR = 4.338, 95% CI 2.397-7.854), involvement of 1/3 of the vagina or not ( OR = 0.497, 95% CI 0.281-0.879), whether topical skin protectants could be used( OR = 3.479, 95% CI 1.596-7.583), whether skin protectants could be used in a standardized manner ( OR = 2.399, 95% CI 1.201-4.789), whether the skin could be cleaned with the correct cleaning agent ( OR = 3.155, 95% CI 1.603-6.209), and whether the skin could be wiped with the correct method ( OR = 3.325, 95% CI 1.806-6.121), totaling 7 predictive factors (all P<0.05). The area under the ROC curve of the prediction model was 0.866, the sensitivity was 0.735, and the specificity was 0.852, the Youden index was 0.587. Model validation results showed the area under the ROC curve was 0.762, the sensitivity was 0.815, the specificity was 0.717, and the accuracy was 80.73%, indicating good predictive performance. Conclusions:The predictive model constructed in this study had good predictive performance and could provide reference for clinical nursing staff to screen high-risk patients with perineal radiation dermatitis.

To understand the infection status of main intestinal protozoa in BALB/c mice and pro-vide a basis for further control of intestinal protozoa infection.Five hundred and forty BALB/c mice provided by four domestic suppliers of BALB/c mice were detected for intestinal protozoa,in which 140 from supplier A,130 from supplier B,135 from supplier C,and 135 from supplier D,re-spectively.Fresh faecal samples were collected from each mouse separately to extract the genome and amplified by nested PCR based on primers for the 18S rRNA gene sequences of Pent-atrichomonas hominis(P.hominis)and Cryptosporidium tyzzeri(C.tyzzeri),and the 16S-like rRNA gene sequence of Tritrichomonas muris(T.muris)and sequenced.The results showed that the total intestinal protozoan infection rate was 7.1％(10/140)in 140 mice faecal samples provided by supplier A.Among them,the positivity rate of T.muris was 7.1％(10/140),C.tyzzeri was 2.1％(3/140),and P.hominis was 7.1％(10/140),the co-infection rate of two intestinal protozoa was 7.1％(10 mice:T.muris+P.hominis),and three intestinal protozoa was 2.1％(3 mice:T.muris+P.hominis+C.tyzzeri).The total intestinal protozoan infection rate in 135 mice faecal samples provided by supplier C was 7.4％,in which,7.4％(10/135)was positive for T.muris.There are no intestinal protozoa to be detected in 130 mice faecal samples from supplier B and 135 mice faecal samples from supplier D.The homology analysis showed that the homology of ampli-fied sequence of T.muris,P.hominis and C.tyzzeri was 98.52％,98.27％and 99.87％compared with published sequence of GenBank No:AY886846.1,GenBank No:AF156964.1 and GenBank No:KJ000486.1,which was clustered as an independent branch by phylogenetic analysis respec-tively.In conclusion,there are intestinal protozoan infection in BALB/c mice in some animal sup-pliers.The co-infections of more than 3 parasites such as T.muris,P.hominis and C.tyzzeri has been found.It will provide a basis for control of intestinal protozoa infection in BALB/c mice in the future.

To prepare a recombinant adeno-associated virus(rAAV9-ZsGreen1-EsASP)capable of expressing the aspartic protease protein of Eimeria stiedai(E.stiedai),and explore its in vitro ac-tivity.The EsASP gene was amplified by PCR,and recombinant adeno-associated viral vector pAAV-IRES-ZsGreen1 was combined with the EsASP gene using homologous recombination to construct the pAAV-ZsGreen1-EsASP expression plasmid;pAAV-ZsGreen1-EsASP expression plasmid,pHelper,and pAAV-RC9 plasmids were cotransfected into HEK-293T cells by liposomal transfection to package and produce recombinant adeno-associated virus rAAV9-ZsGreen1-EsASP capable of expressing EsASP protein.rAAV9-ZsGreen1-EsASP was purified using chloroform treatment-PEG/NaCl precipitation-chloroform extraction method,the purity of the virus was iden-tified by silver staining,the virus morphology was observed by TEM,and virus titer was detected by qRT-PCR;the purified recombinant virus was further infected into HEK-293T cells,and EsASP expression was detected by observing green fluorescent protein ZsGreen1 and Western blot method.The results indicated that double enzyme digestion and DNA sequencing confirmed that the EsASP gene had been successfully constructed into the pAAV-IRES-ZsGreen1 expression plas-mid.The expression of green fluorescent protein in HEK-293T cells suggested that co-transfection was successful.Western blot results of cell protein preparation showed that EsASP protein was successfully expressed;The purified recombinant viral capsid proteins show three distinct bands(VP1-3).The purified rAAV9-ZsGreen1-EsASP was uniform in size,around 20 nm,and had a titer higher than 1.0 × 1012 vg/mL;Green fluorescent protein expression was observed after infection of HEK-293T cells with the recombinant virus,and EsASP expression was detected by Western blot.The results suggest that rAAV9-ZsGreen1-EsASP with in vitro infectious activity was suc-cessfully obtained,providing a material basis for the development of a novel vaccine against Eimer-ia stiedai.

Objective:To construct a risk prediction model for radiation dermatitis in the perineum of cervical cancer patients undergoing radiotherapy, and verify the predictive effect of the model, so as to provide theoretical basis for reducing the risk of perineal radiation dermatitis in clinical practice.Methods:A prospective study design was used to conveniently select 247 cervical cancer patients who received radiotherapy at Tianjin Medical University Cancer Institute & Hospital from January 2022 to March 2023 as the modeling group. They were divided into a group with perineal radiodermatitis ( n = 130) and a group without perineal radiodermatitis ( n = 117). The data of the two groups were compared, and a risk prediction model was constructed based on the results of logistic regression analysis. Used the area under the receiver operating characteristic (ROC) curve to test the predictive performance of the model. From May to November 2023, 109 patients were selected as the validation group to conduct external validation of the model. Results:Among the 247 patients in the modeling group, the age of the group with perineal radiodermatitis was (45.31 ± 7.25) years old, and the age of the group without perineal radiodermatitis was (46.70 ± 6.43) years old. The incidence of perineal radiation dermatitis during radiotherapy in the modeling group was 52.6% (130/247), while in the validation group it was 44.0% (48/109). The final predictive variables included in the model were high blood sugar or not ( OR = 0.304, 95% CI 0.174-0.530), vaginal bleeding/discharge or not ( OR = 4.338, 95% CI 2.397-7.854), involvement of 1/3 of the vagina or not ( OR = 0.497, 95% CI 0.281-0.879), whether topical skin protectants could be used( OR = 3.479, 95% CI 1.596-7.583), whether skin protectants could be used in a standardized manner ( OR = 2.399, 95% CI 1.201-4.789), whether the skin could be cleaned with the correct cleaning agent ( OR = 3.155, 95% CI 1.603-6.209), and whether the skin could be wiped with the correct method ( OR = 3.325, 95% CI 1.806-6.121), totaling 7 predictive factors (all P<0.05). The area under the ROC curve of the prediction model was 0.866, the sensitivity was 0.735, and the specificity was 0.852, the Youden index was 0.587. Model validation results showed the area under the ROC curve was 0.762, the sensitivity was 0.815, the specificity was 0.717, and the accuracy was 80.73%, indicating good predictive performance. Conclusions:The predictive model constructed in this study had good predictive performance and could provide reference for clinical nursing staff to screen high-risk patients with perineal radiation dermatitis.

Objective:To investigate the effects of insulin glargine administration by jet injection versus conventional insulin pen on glucose profile using professional mode flash glucose monitoring(FGM) system in type 2 diabetic patients with poor glucose control.Methods:In this randomized, controlled, crossover study, 40 patients with T2DM who treated with insulin glargine were enrolled. The patients were randomly divided into group A(jet injector-conventional pen, n=20) and group B(conventional pen-jet injector, n=20). Each patient wore FreeStyle Libre sensor from day 4 to day 17. The specialist nurse instructed patients how to master the injection techniques. Professional FGM system was applied to assess glucose profile. Results:The fasting blood glucose(FBG) of the enrolled patients was(9.37±1.84) mmol/L. In contrast to conventional insulin pen, treatment with the jet injector significantly decreased the 24h MBG [(9.06±2.13 vs 9.98±2.67) mmol/L, P=0.001], MaxBG [(16.69±3.01 vs 17.95±3.48) mmol/L, P=0.001], AUC>10 mmol/L [95.93(21.12, 129.02) vs 142.66( 27.88, 198.46), P=0.002], TAR(31.10±21.89 vs 39.49±25.93, P=0.003), MAGE and SDBG. It was observed that patients using jet injector had significant increased TIR(65.94±20.47 vs 58.32±25.00, P=0.001). There were no difference in the risk of hypoglycaemia between two groups. Conclusion:Insulin jet injector was more effective than the insulin pen on glycaemic control and glucose fluctuation without increasing the risk of hypoglycemia in type 2 diabetic patients with uncontrolled glycemia.

OBJECTIVE To investigate the status and effect of influenza vaccination among some elderly population and their cognition of influenza vaccination in Shenzhen Pingshan district， and to provide reference for improving the vaccination willingness of the elderly population. METHODS Descriptive epidemiological method was used to analyze the status quo of influenza vaccination among some elderly populations in Pingshan district； the Logistic regression model was used to analyze the influential factors of influenza vaccination； the serum antibody levels of the elderly population were analyzed before and after influenza vaccination. RESULTS Totally 140 effective survey question naires were collected from elderly. The elderly who participated in the survey had a better cognition of the main transmission mode of the influenza virus， 122 people answered correctly， and the accuracy was 87.1%. Age， knowledge that influenza vaccine should be given once a year and knowledge of Shenzhen’s free vaccination policy were the promoting factors for influenza vaccination behavior among the elderly. The positive rates of the four influenza antibodies （type A H1， type A H3， type B Victoria， type B Yamagata） after inoculation were higher than before inoculation， with statistical significance. The antibody titer levels after vaccination were generally higher than before. CONCLUSIONS The positive rate of antibody testing and antibody titer in the elderly population after influenza vaccination are higher than before， which effectively protects the health of the elderly population. It is necessary to continuously improve the awareness of the elderly population about influenza vaccination and free vaccination policies.

Objective To investigate the benefit finding for caregivers of patients with cancer and its influencing factors. Methods Totally 330 couples of cancer patients and caregivers were investigated from October 2014 to September 2015 by convenience sampling method. The cancer patients were investigated with the Hospital Anxiety and Depression Scale. The caregivers of cancer patients were investigated with Benefit Finding Scale, Zarit Caregiver Burden Interview, Medical Outcomes Study Social Support Survey. Results The total score of benefit finding was (59.65 ± 11.12) points. Regression analysis indicated that caregivers' education, religious, informational and emotional support, caregiver burden, patients' depression symptom, cancer stage and self-caring were influencing factors of benefit finding for caregivers (t=-3.689-5.913, P<0.01 or 0.05), which explained 30.0% of the variance. Conclusions The caregivers of patients with cancer have a low level of benefit finding. Oriented by influencing factors of benefit finding, the health workers should enhance targeted interventions, promote positive perception in the process of caring, develop the quality of caring and life.

Objective Kallikrein 4(KLK4) is a glycosylated,chymotrypsin-like serine protease.KLK4 was first isolated from developing enamel by Japanese scientists,later it was discovered that was expressed in the normal and cancerous tissues.Its main function is to play a role in process of the protein hydrosis,resulting in a normal devel-oping or abnormal pathological changes of the organization.This article reviewed the structure,expression,functions, related disease of KLK4 and other aspects.