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.

Fabry disease, a rare genetic disorder affecting multiple organs, has understudied correlations among enzyme activity, genotype, and clinical manifestations in patients of different sexes with classical and late-onset phenotypes. In this study, clinical data, α-Gal A activity, and GLA gene test results of 311 patients, who were categorized by classical and late-onset phenotypes, ⩽5% and > 5% of the normal mean value of enzyme activity, and truncated and nontruncated mutation groups, were collected. The common clinical manifestations of Fabry disease included acroparesthesia, hypohidrosis/anhidrosis, neuropsychiatric system, and renal and cardiovascular involvement. Multiorgan involvement was higher in males and classical phenotype patients. In both sexes, classical patients commonly presented with acroparesthesia and multiorgan involvement, whereas late-onset patients showed renal, neuropsychiatric, and cardiovascular involvement. Male and classical patients had lower enzyme activity than female and late-onset patients, respectively. Classical males with enzyme activity of ⩽5% of the normal mean level showed higher multiorgan involvement frequency than those with enzyme activity of > 5%, whereas no significant difference was observed among females. Ninety-five gene mutation sites were detected, with significant phenotype heterogeneity in patients with the same mutation. No significant difference in enzyme activity or clinical manifestations was observed between truncated and nontruncated mutations. Overall, male patients with Fabry disease, regardless of classical or late-onset phenotype, have a higher frequency of multiple-organ involvement and lower α-Gal A activity than female patients. α-Gal A activity was closely correlated with clinical symptoms in males but weakly correlated with clinical manifestations in females. The clinical manifestations of patients with the same mutation are heterogeneous, and the correlation between gene mutation and enzyme activity or clinical manifestation is weak.
Adolescent ; Adult ; Aged ; Female ; Humans ; Male ; Middle Aged ; Young Adult ; Age of Onset ; alpha-Galactosidase/metabolism* ; China ; Fabry Disease/enzymology* ; Genotype ; Mutation ; Phenotype ; Sex Factors ; East Asian People/genetics*

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.

The "pre-disease" theory of traditional Chinese medicine focuses on the dynamic and continuous evolution from health to disease, and emphasizes early identification and intervention in the complex and gradual process of evolution from health to disease. The "pre-disease" theory and complexity science share the same perspective on health and disease from the standpoint of features of the dynamic evolution and holism, i. e., life is considered as a complex system with ongoing dynamic changes, which exhibit the nonlinear features of " homeostasis-destabilization-phase transition-another homeostasis". In this paper, from the perspective of nonlinear dynamics in complexity science, we explain the scientific connotation of the evolution law of "pre-disease"-disease based on the theory of critical slowing down in traditional Chinese medicine. Based on the theory of critical slowing down and the dynamic network biomarker method generated by its development, combined with the macro signs of comprehensive analysis of data gained by four diagnostic method and the micro features including transcriptomics and the microbiomics, this paper proposes to integrate macro and micro multi-hierarchy information to construct a "pre-disease" critical slowing down identification model with the characteristics of traditional Chinese medicine diagnosis and treatment, which provides a new perspective and method for the early warning of complex diseases.

OBJECTIVE To determine the prevalence of frontal recess cells variations in patients with frontal sinus associated headache according to the International Frontal Sinus Anatomy Classification(IFAC).METHODS A retrospective study was conducted on the CT scans of sinuses in patients with frontal sinus associated headache.We reviewed 46 patients with frontal sinus-related headache who had clinical symptoms and were relieved after nasal endoscopic surgery.The development of frontal recess cells in the frontal recess drainage area was analyzed,and the variation of middle meatus and sinus involvement were analyzed in the same time.The Anatomical variations and imaging characteristics of frontal recess cells development in patients with frontal sinus associated headache were analyzed.RESULTS A total of 92 sinus CT profiles were analyzed in 46 patients.The most common cells were agger nasi cell(ANC)(100%,92/92),followed by supra bulla cell(SBC)(78.3%,72/92),supra agger cell(SAC)(67.4%,62/92),supra bulla frontal cell(SBFC)(27.2%,25/92),supra agger frontal cell(SAFC)(20.7%,19/92),frontal septal cell(FSC)(8.7%,8/92)and supraorbital ethmoid cell(SOEC)(0%,0/92).In the conventional frontal sinus drainage area,SAFC(P=0.0108),SAC(P=0.0104)and SAFC(P=0.0088)in the IFAC classification were significantly associated with the occurrence of frontal sinus associated headache.At the same time,the middle concha bullosa also showed a significant correlation with the occurrence of frontal sinus associated headache in the lower segment of the frontal recess drainage channel(P=0.0390).CONCLUSION In the frontal recess drainage channel,the abnormal development of SAC,SAFC,SBFC and the middle concha bullosa are significantly correlated with frontal sinus associated headache.

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.

The meniscus in the knee joint plays an important role in bearing load,shock absorption,joint lubrication,and increasing the stability of the knee joint in various directions.Meniscal injury is a common ailment in knee joint injuries,which can cause severe knee pain and limited knee joint movement,significantly affecting the patient's quality of life.Arthroscopic minimally invasive treatment has become the main approach for treating meniscal injuries,with the consensus being to preserve the meniscus morphology and structure as much as possible.By introducing the structure and function of the meniscus,the mechanism of injury,and focusing on several suture techniques for arthroscopic meniscal injury treatment,analyzing their advantages and disadvantages,it aims to provide some reference for clinical research on arthroscopic suture of the meniscus.

Heme, which exists widely in living organisms, is a porphyrin compound with a variety of physiological functions. Bacillus amyloliquefaciens is an important industrial strain with the characteristics of easy cultivation and strong ability for expression and secretion of proteins. In order to screen the optimal starting strain for heme synthesis, the laboratory preserved strains were screened with and without addition of 5-aminolevulinic acid (ALA). There was no significant difference in the heme production of strains BA, BAΔ6 and BAΔ6ΔsigF. However, upon addition of ALA, the heme titer and specific heme production of strain BAΔ6ΔsigF were the highest, reaching 200.77 μmol/L and 615.70 μmol/(L·g DCW), respectively. Subsequently, the hemX gene (encoding the cytochrome assembly protein HemX) of strain BAΔ6ΔsigF was knocked out to explore its role in heme synthesis. It was found that the fermentation broth of the knockout strain turned red, while the growth was not significantly affected. The highest ALA concentration in flask fermentation reached 82.13 mg/L at 12 h, which was slightly higher than that of the control 75.11 mg/L. When ALA was not added, the heme titer and specific heme production were 1.99 times and 1.45 times that of the control, respectively. After adding ALA, the heme titer and specific heme production were 2.08 times and 1.72 times higher than that of the control, respectively. Real-time quantitative fluorescent PCR showed that the expressions of hemA, hemL, hemB, hemC, hemD, and hemQ genes at transcription level were up-regulated. We demonstrated that deletion of hemX gene can improve the production of heme, which may facilitate future development of heme-producing strain.
Gene Deletion ; Bacillus amyloliquefaciens/metabolism* ; Aminolevulinic Acid/metabolism* ; Heme/metabolism* ; Fermentation

Purpose: To explore the effect of intravesical electrical stimulation (IVES) on urinary adenosine triphosphate (ATP) and nitric oxide (NO) in rats with detrusor underactivity (DU) induced by bilateral pelvic nerve crush (bPNC), and to determine the underlying peripheral mechanism. Methods: Twenty-four female Sprague-Dawley rats were equally divided into 3 groups: sham; bPNC; and IVES. Rats in the IVES group began to receive IVES treatment 10 days after bPNC (20 minutes per day for 14 consecutive days). After the 14th IVES, rat urine was collected and cystometry was performed. The serum creatinine, blood urea nitrogen, and urinary ATP and NO levels were measured, and a routine urinalysis was performed. Results: The maximum cystometric capacity (MCC), maximum changes in bladder pressure during filling (∆FP), and postvoid residual urine (PVR) in the IVES group were significantly lower than the bPNC group, and the maximum changes in bladder pressure during voiding (∆VP) was significantly higher than the bPNC group. Compared with the sham group, the MCC, ∆FP and PVR were significantly increased, and the maximum voiding pressure (MVP) and ∆VP were significantly decreased in the bPNC group. After bPNC, urinary ATP was significantly decreased, and urinary NO was significantly increased. In IVES-treated rats, urinary ATP was significantly higher than the bPNC group, and NO was significantly lower than the bPNC group. In addition, the ATP-to-NO ratio of the rats in the bPNC group was significantly lower than the sham and IVES groups. Correlation analysis showed that the ATP and NO were not correlated with the MCC, ∆FP, MVP, ∆VP, and PVR. Conclusions Promoting the release of urothelial ATP and inhibiting the release of urothelial NO may be one of the peripheral mechanisms underlying IVES in the treatment of DU. Specifically, IVES may shift the balance between excitation and inhibition toward excitation.