ObjectiveThe U6 promoter is an essential element for driving sgRNA expression in the clustered regularly interspaced short palindromic repeat sequences/CRISPR-associated protein 9（CRISPR/Cas9）gene editing system in dicotyledonous plants. Endogenous U6 promoters typically exhibit higher transcriptional activity， which can significantly improve gene editing efficiency. This study aims to identify endogenous U6 promoters in Artemisia annua to optimize its CRISPR/Cas9 gene editing system， which holds significant importance for its molecular breeding. MethodsOn the basis of the highly conserved U6 snRNA sequences in Arabidopsis thaliana， endogenous U6 promoters were screened in the A. annua genome. Expression vectors were constructed with candidate AaU6 promoter driving the firefly luciferase （LUC） reporter gene， and then transiently transformed into Nicotiana benthamiana. Transcriptional activities of the promoters were measured and compared by in vivo imaging and the Dual Luciferase Reporter assay. ResultsEight endogenous U6 promoters were successfully cloned from A. annua. Sequences alignment revealed that all these promoters contained the two conserved cis-acting elements， upstream sequence element （USE） and TATA-box， which affected their transcriptional activity. Dual-luciferase activity assays indicated that the transcriptional activities of AaU6-3， AaU6-1， and AaU6-5 were significantly higher than that of the Arabidopsis AtU6-26 promoter， with AaU6-3 exhibiting the highest activity. ConclusionThis study identified three endogenous AaU6 promoters with high transcriptional activity in A. annua， providing key functional elements for establishing an efficient gene editing system in A. annua. These findings will contribute to advancing precision molecular breeding and high-quality germplasm innovation in A. annua.

ObJective·To evaluate the dentofacial phenotype in non-syndromic tooth agenesis(NSTA)patients with paired box gene 9(PAX9)mutation.Methods·Patients with NSTA who visited the Department of Second Dental Center of Shanghai Ninth People's Hospital,Shanghai Jiao Tong University School of Medicine,between January 2016 and December 2023 received whole-exome sequencing to screen PAX9 mutation.The location and number of missing teeth were evaluated by oral pantomography,and dentofacial deformities were evaluated by X-ray cephalometrics.Results·Seven patients with PAX9 mutation were included in the study,including 3 males(42.9％)and 4 females(57.1％).The patients were 7-31 years old at first visit,with a mean age of(19.7±8.0)years old.All the 7 patients were PAX9 heterozygotes,of which 4 were missense and 3 were frameshift.The average number of missing teeth was 15.9±2.9.The number of missing teeth in maxilla(9.6±2.6)was slightly higher than that in mandible(6.3±2.4)(P=0.030).Maxillary second molar(100.0％),maxillary canine(85.7％)and mandibular second premolar(85.7％)were the three most common missing teeth,while mandibular lateral incisor(14.3％)and mandibular canine(14.3％)were the two least missing teeth.Patients with frameshift mutation had more missing teeth(18.3±2.1)than those with missense mutation(14.0±1.8)(P=0.032).X-ray cephalometrics analysis results showed that the angle sella-nasion-subspinale(SNA),angle nasion-subspinale-subspinale-porion(NA-Apo)and sella-nasion(S-N)in adult patients with PAX9 mutation were significantly lower than the normal reference values,suggesting a shorter anterior cranial base and maxillary length.The frankfort horizontal plane-nasion-porion(FH-NPo)was higher than the reference value,and the Y-axis was lower than the reference value,indicating a more prognathic mandible.The angle subspinale-nasion-supramental(ANB)was lower than the reference value,indicating a skeletal angle Ⅲ malocclusion.The angle upper central incisor-nasion-subspinale(angle U1-NA)was higher than the reference value,indicating a lip inclination of maxillary central incisor.The angle lower central incisor-mandibular plane(IMPA)and lower central incisor-nasion-supramental(L1-NB)were lower than the reference values,indicating a retroclination of the mandibular central incisor,and crossbite in the maxillary and mandibular anterior teeth.Conclusion·The dentofacial phenotype of PAX9-mutated patients with NSTA is reported comprehensively.It is helpful to improve the understanding of the role of PAX9 in human maxillofacial development.

Objective To understand the awareness level of iodine deficiency (ID) impairments and the attitude on edible iodized salt,and its consumption among doctors and nurses in cities,in order to provide scientific evidence for health education on iodine deficient disorders (IDD) in the future.Methods The questionnaire was self-designed,and spread through the web page of Wenjuanxing,an online server company,from May 6 to June 6,2017.At the end of the survey,a total of 481 valid questionnaires were reclaimed,in which ratio of gender was female 63.8％ (307/481),male 36.2％ (174/481).The data were analyzed by SPSS 22.0,including logistic regression analysis with backward according to the statistical significant level of P ＜ 0.05.Results Among doctors and nurses,90.4％ (435/481) knew ID impairments;72.8％ (350/481) answered intelligent disability as the most serious problem of ID;55.5％ (267/481)misunderstood that the areas in which they lived were not ID areas,although all cities surveyed were ID areas;41.0％ (197/481) of doctors and nurses misunderstood that coastal residents did not need to consume iodized salt.About the evaluation of iodine nutrition status of current population,15.6％ (75/481) of respondents judged as iodine excess.About consuming edible salt,76.3％ (367/481) selected iodized,9.6％ (46/481)non-iodized,and 14.1％ (68/481) both iodized and non-iodized.The results of logistic regression analysis showed the factors that prevents health care workers from choosing iodized salt were:"living in coastal areas","think him or her as iodine adequate","know that iodine deficiency can affect the development of children but still adhere to the consumption of non-iodized salt";the factors that promoted the choice of iodized salt for medical staff were "insist on buying iodized salt,and do not choose non-iodized salt",and 75.8％ (238/314)of them knew that intelligent disability as the most serious problem of ID.Conclusions Most doctors and nurses have high level of knowledge on ID and its control.But lack of information,as well as misunderstanding of "coastal areas iodine adequate" and worry about "excess iodine causes thyroid diseases and cancer",which would hinder the active consumption of iodized salt.Health education for them should be conducted through professional ways,stressing on the threaten of ID environment,sharing the information about national and local progress on control of IDD and iodized salt safety,and clarifying the relationship between iodine salt or iodine and thyroid cancer and nodules.

Piperazine drugs, N - benzyl piperazine (BZP), 1 - (3 - chlorobenzene) piperazine (mCPP), 1 - (3 - three lfuorinated methyl phenyl) piperazine (TFMPP) have excitation and hallucinogenic effects similar to MDMA. They have became the alternative LSDs, abuse of substitues in the world reported increased year by year. The paper reviewed three piperazines regulation, abuse, toxicology, detection methods, and hope to provide a reference for judicial departments to crack down on the crime of the drug uses.

BACKGROUND: Uncoupling of major histocompatibility complex (MHC) is the main cause for transplantation rejection, and it is the best way to prevent transplantation rejection by induce immunological tolerance of the recipient to the donor organ. Self-tolerant T cells can be obtained by negative selection in thymus, whether the intrathymic injection of allogenic antigen can get the immunological tolerance to the antigen?OBJECTIVE: To observe the effects of intrathymic injection of allogenic antigen on inducing immunological tolerance in nerve transplantation.DESIGN: A comparative observation.SETTINGS: Department of Immunology, Harbin Medical University; Department of Orthopaedics, Fourth Clinical Hospital of Harbin Medical University.MATERIALS: Thirty donor C57BL/6 mice (H-2b), male, aged from 6-8 weeks, weighing 18-22 g, were purchased from the Veterinarian Institute of Heilongjiang Province; While 60 recipient Balb/c mice (H-2b) female, aged from 6-8 weeks, weighing 18-22 g, from Beijing Experimental Animal Center. MHC (H-2b) antigen was prepared by the Department of Immunology, Harbin Medical University, and the concentration of protein was 4.4 g/L.METHODS: The experiments were carried out in the Department of Immunology, Harbin Medical University from June to November 2002. The recipient Balb/c mice were randomly divided into four groups: intrathymic injection group, syngenic transplantation group, allogenic transplantation group and immunosuppressant drug group. MHC (H-2b) antigen was extracted from splenic cells of donor C57BL/6 mice and injected intrathymically into recipient Balb/c mice (H-2d). Two weeks later, the sciatic nerve was transplanted to the recipient mice. Mixed lymphocyte reaction and delayed-type hypersensitivity (DTH) were detected at 3 weeks after transplantation.MAIN OUTCOME MEASURES:The differences of mixed lymphocyte reaction and DTH were compared among the groups.RESULTS: All the 30 donor C25BL/6 mice (H-2b) and 60 recipient Balb/c mice (H-2d) were involved in the analysis of results.①Results of mixed lymphocyte reaction: The cell proliferations in the syngenic transplantation group and intrathymic injection group were obviously lower than that in the allogenic transplantation group [(546.1±75.1), (2 668.3±533.8), (3 101.3±429.1), (4 312.3±534.1) minutes-1, P＜0.05].②Results of DTH: The thicknesses difference between two pads in the syngenic transplantation group and intrathymic injection group were obviously lower than that in the allogenic transplantation group [(41.1±3.7), (72.1±5.1), (57.6±11.3), (86.2±13.2)μm, P＜0.05].CONCLUSION:The intrathymic injection of donor H-2b antigen could induce immunological tolerance of nerve transplantation.