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.

Fascia,the initial response site for mechanical stimulation in manipulations,is also the target of the effect of manipulations.As the essence of manipulation is"force",how mechanical stimuli are transduced into neuroelectric and biochemical signals in the fascia and how physical and chemical signals of the fascia initiate the mechanical stimulation effect are the common key questions in the study of the principle of manipulation.The physical changes in the fascial connective tissue caused by the manipulation,such as the deformation and displacement of the fascial tissue,can act on the nerve end receptors in the fascial layer and generate neural electrical signals;they can also activate the mechanoreceptors on the fascial cell membrane and convert mechanical signals into chemical signals via mechanosensitive ion channel transduction,triggering a physicochemical coupling response in the fascial microenvironment and producing mechanical stimulation through neuro-endocrine-immune system pathways.The"mechanical force of manipulation"in the fascia is transmitted through the meridian to facilitate the body's perception and transmission of mechanical stimulation signals,indicating that the fascia is the"sensor"of coupled response to the physicochemical information of mechanical stimulation of manipulation.

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 explore EDAR(ectodysplasin A receptor)gene variants that lead to congenital tooth agenesis,and preliminarily analyze the reasons why variants in EDAR can cause both syndromic and non-syndromic tooth agenesis.Methods·Patients with congenital tooth agenesis admitted to the Department of 2nd Dental Center,Shanghai Ninth People's Hospital,Shanghai Jiao Tong University School of Medicine and their family members were included,and genomic DNA from their peripheral blood was extracted for whole exome sequencing(WES).After preliminary screening,PolyPhen-2,Mutation Taster,and Provean were used to predict the harmfulness of potential variants.The screened variants in patients and their families were verified by Sanger sequencing.Conservation analysis of variants was performed,and Swiss-Model was used to analyze the changes in the three-dimensional structure of EDAR.The teeth and syndromic phenotype of the patients and their family members were investigated.Results·Among the included congenital tooth agenesis patients,five patients with EDAR mutations were found,one with EDAR frameshift mutation c.368_369insC(p.L123fs)and the other four with EDAR missense mutations.Two of these four patients were diagnosed as non-syndromic tooth agenesis(NSTA),resulted from c.77C＞A(p.A26E)homozygous mutation and c.380C＞T(p.P127L)heterozygous mutation,respectively.The other two patients with two variants were diagnosed as hypohidrotic ectodermal dysplasia(HED).One compound heterozygous missense mutation patient carried EDAR c.77C＞T(p.A26V)from her father andEDAR c.1281G＞C(p.L427F)from her mother;the other patient with both EDAR and EDA mutations carried EDAR c.1138A＞C(p.S380R)heterozygous mutation and EDA c.1013C＞T(p.T338M)hemizygous mutation.Both variants were from his mother and were reported to be related with NSTA.Two of these missense mutations,EDAR c.1281G＞C(p.L427F)and EDAR c.77C＞A(p.A26E),had not been reported before.The missense mutations affected the protein's spatial conformation by altering the polarity,charge,or volume of the amino acid residues.The frameshift mutation caused a non-triplet base addition,which probably led to protein truncation or degradation.Conclusion·Two new EDAR missense mutations are discovered.An NSTA patients with EDAR homozygous mutations and an HED patient with both EDA and EDAR mutations are reported.It expands the understanding of pathogenic mechanisms of EDAR mutations causing HED and NSTA.

Objective:Evaluate the influence of different pressure transmission media of urodynamic water filled catheter(WFC) and air charged catheter(ACC) on the pressure measurement results to determine whether they can be used interchangeably.Methods:The results of 2 147 patients who underwent urodynamic examination in our hospital from January 2014 to December 2020 were retrospectively analyzed. A total of 2 538 times of bladder manometry data were obtained, including 1 299 times in WFC group, 856 times in male and 443 times in female, aged 37(24, 50)years, course of disease 1.2(0.4, 5.0) years, 1 130 times in neurogenic bladder(NB)and 169 times in non-neurogenic bladder(N-NB); In ACC group, there were 1 239 times, 773 times for male and 466 times for female, with age of 37(24, 55)years, course of disease of 1.5(0.5, 6.0)years, 1 040 times for Nb and 199 times for N-NB. There was no significant difference in baseline data of general clinical data between the two groups. The intravesical pressure(Pves), intra-abdominal pressure(Pabd)and detrusor pressure(Pdet) of WFC and ACC patients during filling and urination were analyzed. For traumatic spinal cord injury(SCI) and idiopathic patients, the two sets of pressure measurement data were analyzed separately. Nonparametric test and Chi-square test were used to compare the Pves, Pabd, and Pdet recorded by the two manometry catheters before, at the end and after urination, the maximum detrusor pressure at DO(Pdet.max-DO), and the maximum detrusor pressure during spontaneous urination (Pdet. max) and the detrusor pressure (Pdet.Qmax) corresponding to the maximum urine flow rate, the maximum urethral pressure (MUP) and the maximum urethral closure pressure (MUCP) during resting urethral pressure profile, and the initial cough Pdet signal pattern (typeⅠ, typeⅡand typeⅢ).Results:Regardless of the cause, the Pabd values measured by ACC were significantly higher than WFC before filling, end filling and after voiding[18(10, 26)cmH 2O vs.15(11, 21)cmH 2O; 23(16, 31)cmH 2O vs. 20(14, 26)cmH 2O; 23(15, 31)cmH 2O vs.18(12, 24)cmH 2O], and Pdet were significantly lower than WFC[0(0, 0) cmH 2O vs. 0(0, 1)cmH 2O; 5(1, 13)cmH 2O vs. 9(4, 17)cmH 2O; 6(1, 12)cmH 2O vs. 7(3, 14)cmH 2O]. In the initial cough state, Pves and Pabd increase value were also significantly lower than that of WFC [22(12, 36)cmH 2O vs. 23(14, 38)cmH 2O; 20(10, 33)cmH 2O vs. 21(12, 36)cmH 2O]. The Pves measured by ACC was also significantly higher than WFC before filling and after voiding[18(10, 27)cmH 2O vs. 16(11, 21)cmH 2O; 30(22, 39)cmH 2O vs. 26(20, 36)cmH 2O]. Maximum urethral pressure (MUP) and maximum urethral closure pressure (MUCP) measured by ACC were significantly higher than WFC [91(69, 118)cmH 2O vs.81(64, 106)cmH 2O; 77(55, 103)cmH 2O vs. 68(48, 91)cmH 2O], and there were no significant differences in Pdet.max-DO、Pdet.max和Pdet.Qmax. For patients with traumatic SCI, the Pves measured by ACC was significantly higher than WFC before filling[15(10, 24)cmH 2O vs. 14(10, 20)cmH 2O], and only MUP was significantly higher than WFC in the measurement of urethral pressure[95(71, 119)cmH 2O vs. 85(65, 112)cmH 2O], and there were no significant differences in Pdet.max-DO, Pdet.max, Pdet.Qmax and MUCP. For idiopathic patients, Pves measured by ACC before filling and after urination were significantly higher than WFC[25(20, 29)cmH 2O vs. 18(11, 23)cmH 2O; 35(29, 44)cmH 2O vs. 28(20, 38)cmH 2O], while Pdet.max-DO, Pdet.max, Pdet.Q max, MUP and MUCP were not significantly different in different pressure measurement systems. For the comparison of the initial cough Pdet signal pattern, ACC is easier to detect type Ⅰ, and WFC is easier to detect type Ⅱ and type Ⅲ. Conclusions:Compared with WFC, ACC measured higher Pves and Pabd and lower Pdet in resting state, and lower Pves and Pabd in initial cough state. The pressure values and signal pattern measured by WFC and ACC are not completely consistent, so they cannot be used interchangeably.

Objective:To observe the effect of noninvasive positive pressure ventilation (NIPPV) and high-flow nasal cannula oxygen therapy (HFNC) on the prognosis of patients with coronavirus disease 2019 (COVID-19) accompanied with acute respiratory distress syndrome (ARDS).Methods:A retrospective study was conducted in Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology when authors worked as medical team members for treating COVID-19. COVID-19 patients with pulse oxygen saturation/fraction of inspiration oxygen (SpO 2/FiO 2, S/F) ratio < 235, managed by medical teams [using S/F ratio instead of oxygenation index (PaO 2/FiO 2) to diagnose ARDS] from February to April 2020 were included. The patients were divided into NIPPV group and HFNC group according to their oxygen therapy modes. Clinical data of patients were collected, including general characteristics, respiratory rate (RR), fraction of FiO 2, SpO 2, heart rate (HR), mean arterial pressure (MAP), S/F ratio in the first 72 hours, lymphocyte count (LYM), percentage of lymphocyte (LYM%) and white blood cell count (WBC) at admission and discharge or death, the duration of dyspnea before NIPPV and HFNC, and the length from onset to admission. The differences of intubation rate, all-cause mortality, S/F ratio and RR were analyzed, and single factor analysis and generalized estimation equation (GEE) were used to analyze the risk factors affecting S/F ratio. Results:Among the 41 patients, the proportion of males was high (68.3%, 28 cases), the median age was 68 (58-74) years old, 28 cases had complications (68.3%), and 34 cases had multiple organ dysfunction syndrome (MODS, 82.9%). Compared with HFNC group, the proportion of complications in NIPPV group was higher [87.5% (21/24) vs. 41.2% (7/17), P < 0.05], and the value of LYM% was lower [5.3% (3.4%-7.8%) vs. 10.0% (3.9%-19.7%), P < 0.05], the need of blood purification was also significantly lower [0% (0/24) vs. 29.4% (5/17), P < 0.05]. The S/F ratio of NIPPV group gradually increased after 2 hours treatment and RR gradually decreased with over time, S/F ratio decreased and RR increased in HFNC group compared with baseline, but there was no significant difference in S/F ratio between the two groups at each time point. RR in NIPPV group was significantly higher than that in HFNC group after 2 hours treatment [time/min: 30 (27-33) vs. 24 (21-27), P < 0.05]. There was no significant difference in rate need intubation and hospital mortality between NIPPV group and HFNC group [66.7% (16/24) vs. 70.6% (12/17), 58.3% (14/24) vs. 52.9% (9/17), both P > 0.05]. Analysis of the factors affecting the S/Fratio in the course of oxygen therapy showed that the oxygen therapy mode and the course of illness at admission were the factors affecting the S/F ratio of patients [ β values were -15.827, 1.202, 95% confidence interval (95% CI) were -29.102 to -2.552 and 0.247-2.156, P values were 0.019 and 0.014, respectively]. Conclusion:Compared with HFNC, NIPPV doesn't significantly reduce the intubation rate and mortality of patients with COVID-19 accompanied with ARDS, but it significantly increases the S/F ratio of those patients.

Objective:To evaluate the clinical efficacy of tendon-regulating manipulation plus kinesiotherapy in treating low back pain. Methods:Sixty patients were randomized into a treatment group and a control group by using the random number table, 30 cases in each group. The treatment group was intervened by tendon-regulating manipulation plus kinesiotherapy, while the control group was by the tendon-regulating manipulation alone. The lumbar lordosis was measured by X-ray (side view), the pain was evaluated by analgesy meter, the lumbar range of motion was by using goniometer, and the function was judged by Oswestry disability index (ODI) before and after treatment, and the therapeutic efficacy was also observed. Results:After treatment, the pain level was significantly reduced, lumbar lordosis was significantly increased, the lumbar range of motion was markedly improved, and the ODI score significantly dropped (allP<0.05) in both groups; the improvement of each item in the treatment group was more significant than that in the control group (allP<0.05). The total effective rate was 90.0% in the treatment group versus 63.3% in the control group, and the difference was statistically significant (P<0.05). Conclusion:In the treatment of low back pain, tendon-regulating manipulation plus kinesiotherapy can mitigate topical pain, improve the motion of low back, enhance the quality of life, and produce a more significant therapeutic efficacy compared to tendon-regulating manipulation alone.

Objective To investigate the variation of procalcitonin(PCT) in blood and tissue level of acute pancreatitis rats and probe its significant. Methods One hundred and two male Wistar rats were randomly divided into control group ( n = 6 ), lipopolysaccharide group ( LPS, n = 24 ), acute edematous pancreatitis (AEP) group ( n = 24), acute necrotizing pancreatitis (ANP) group ( n = 24), AN P + LPS group ( n = 24). Subcutaneous injection of cerulein was used for AEP induction, while ANP model was induced by retrograde injection of sodium taurocholate into the biliary and pancreatic duct. The rats were sacrificed at 3,6, 18 and 24 hours after model induction. Pancreatic tissue was harvested and the pathological scores were assessed. Levels of PCT in serum, liver, lung, spleen, pancreas, small intestine, large intestine tissue was harvested and tissue levels of PCT were determined. Results AEP and ANP models were established successfully. At 6 h, the serum levels of PCT in control group, LPS group, AEP group, ANP group and ANP +LPS group were (0.0144 ±0.0082) ng/ml, (0. 1722 ±0.0449) ng/ml,(0.4751 ±0.0572) ng/ml, (0.7070 ±0. 1040) ng/ml and ( 1. 1960 ±0.8644) ng/ml, respectively; and the difference was statistically significant (P < 0.05 ). PCT could be detected in liver, lung, spleen, pancreas, small intestine and large intestine tissue of normal rats. PCT levels in liver and pancreas of ANP group were not statistically different, but the PCT levels in lung, spleen, and large intestine tissue significantly decreased, and the corresponding values were (5.63 ±0.62) ng/ml vs. (6.85 ±0.46) mg/ml, (4.73 ±1.27) mg/ml vs. (6.88 ±0.37) ng/ml, (1.08 ±0.52) ng/ml vs. (4.12 ± 1.02) ng/ml (P <0.01 ). However, the PCT levels in small intestine significantly increased, which were (2.51 ±0.90) ng/ml vs (0.98 ±0. 12) ng/ml (P<0. 01). Conclusions Serum PCT level was associated with the severity of AP and infection; the changes of PCT levels in different tissues may be related with the changes of organ's function.