AIM:To investigate the mechanism by which muscle segment homeobox 2(Msx2)regulates the differentiation of outer enamel epithelial cells in the enamel organ.METHODS:Tissue paraffin sections were prepared and subjected to hematoxylin-eosin(HE)staining to analyze the effect of Msx2 deficiency on the differentiation status of epithelial cells in the enamel organ at the morphological level.At the ultrastructural level,alterations in cell structure were analyzed.The intermediate steps mediating cell differentiation were identified.Transcriptome sequencing analysis was performed to validate the molecular mechanisms underlying the observed phenomena.RESULTS:Msx2 deficiency was innovatively found to induce severe squamous epithelial hyperplasia in outer enamel epithelial cells of enamel organ,accompanied by dynamic restructuring of the cell cytoskeleton and alterations in cell adhesion at the ultrastructure level.As a transcriptional repressor,the loss of Msx2 expression results in significant increases(P＜0.05 or P＜0.01)in the mRNA expression levels of integrin β2(Itgβ2),ItgαM,Itgα4,Rac family small GTPase 2(Rac2),Rac/Cdc42 guanine nucleo-tide exchange factor 6(Arhgef6)and protein tyrosine phosphatase receptor type C(Ptprc).CONCLUSION:Msx2 regu-lates cytoskeleton structure and cell-cell interaction through the Rho GTPases signaling pathway,thereby influencing the differentiation state of outer enamel epithelial cells.This study reveals the mechanism through which Msx2 regulates the differentiation of outer enamel epithelial cells,providing a theoretical foundation for the prevention and treatment of enam-el-related clinical dental diseases.

Atherosclerosis(AS)is a chronic inflammatory disease involving disorders of lipid and iron metabolism.The establishment of suitable animal models is required to further the study of the etiology,pathogenesis,prevention,and therapeutic measures of AS.The main animal models of AS related to iron and lipid metabolism are mice and miniature piglets,especially male ApoE-/-mice.Single-factor high-fat diet-induced iron and lipid metabolism disorders are a common type of AS model,manifesting as elevated blood lipid levels,large plaques and iron deposition in the aorta,and significant increases in serum and liver iron levels.This review compares the effects of different intervention factors on iron and lipid metabolism in AS animal models,and summarizes the method of establishing AS animal models using dietary induction,chemical intervention,and gene modification,to provide references and inspiration for future research into AS and metabolic diseases and the development of new drugs.

AIM:To investigate the mechanism by which muscle segment homeobox 2(Msx2)regulates the differentiation of outer enamel epithelial cells in the enamel organ.METHODS:Tissue paraffin sections were prepared and subjected to hematoxylin-eosin(HE)staining to analyze the effect of Msx2 deficiency on the differentiation status of epithelial cells in the enamel organ at the morphological level.At the ultrastructural level,alterations in cell structure were analyzed.The intermediate steps mediating cell differentiation were identified.Transcriptome sequencing analysis was performed to validate the molecular mechanisms underlying the observed phenomena.RESULTS:Msx2 deficiency was innovatively found to induce severe squamous epithelial hyperplasia in outer enamel epithelial cells of enamel organ,accompanied by dynamic restructuring of the cell cytoskeleton and alterations in cell adhesion at the ultrastructure level.As a transcriptional repressor,the loss of Msx2 expression results in significant increases(P＜0.05 or P＜0.01)in the mRNA expression levels of integrin β2(Itgβ2),ItgαM,Itgα4,Rac family small GTPase 2(Rac2),Rac/Cdc42 guanine nucleo-tide exchange factor 6(Arhgef6)and protein tyrosine phosphatase receptor type C(Ptprc).CONCLUSION:Msx2 regu-lates cytoskeleton structure and cell-cell interaction through the Rho GTPases signaling pathway,thereby influencing the differentiation state of outer enamel epithelial cells.This study reveals the mechanism through which Msx2 regulates the differentiation of outer enamel epithelial cells,providing a theoretical foundation for the prevention and treatment of enam-el-related clinical dental diseases.

Atherosclerosis(AS)is a chronic inflammatory disease involving disorders of lipid and iron metabolism.The establishment of suitable animal models is required to further the study of the etiology,pathogenesis,prevention,and therapeutic measures of AS.The main animal models of AS related to iron and lipid metabolism are mice and miniature piglets,especially male ApoE-/-mice.Single-factor high-fat diet-induced iron and lipid metabolism disorders are a common type of AS model,manifesting as elevated blood lipid levels,large plaques and iron deposition in the aorta,and significant increases in serum and liver iron levels.This review compares the effects of different intervention factors on iron and lipid metabolism in AS animal models,and summarizes the method of establishing AS animal models using dietary induction,chemical intervention,and gene modification,to provide references and inspiration for future research into AS and metabolic diseases and the development of new drugs.

The classic formula Wuyaotang is the 49^th of the 100 formulas in the Catalogue of Ancient Classic Prescriptions (First Batch) issued by the National Administration of Traditional Chinese Medicine, and is from the Secrets from the Orchid Chamber (《兰室秘藏》) by LI Dongyuan of the Jin Dynasty. It is composed of Angelicae Sinensis Radix, Glycyrrhizae Radix et Rhizoma, Aucklandiae Radix, Linderae Radix, and Cyperi Rhizoma, and has the effect of moving Qi, regulating meridians, and relieving pain. It is mainly indicated for Qi stagnation and blood stasis syndrome. Based on the ancient books on Wuyaotang, this study systematically reviewed the formula source, composition, dosage, preparation, usage, functions, indications, preparation principle, drug processing, modification, etc. of Wuyaotang with the bibliometrics method, explored its historical evolution, and determined the key information. Statistical analysis of its modern literature shows that there are few studies of the original formula of Wuyaotang, and the clinical studies mainly focus on modified Wuyaotang. It has a wide range of treatment scope and can be used for the treatment of dysmenorrhea, delayed menstrual cycle, hypomenorrhea, and menstrual fever, as well as ulcerative colitis, spleen distortion, sciatica, child intestinal spasm, and other internal, surgical, gynecological, and pediatric diseases. The pathogenesis in traditional Chinese medicine (TCM) is Qi stagnation. Through the analysis and research on ancient books and modern literature recording Wuyaotang, this study is expected to provide a scientific basis for the clinical application, in-depth research, and development of the classic formula Wuyaotang.

Objective:To clarify the clinical characteristics and related fators of children with delayed antibody production of mycoplasma pneumoniae pneumonia(MPP).Methods:Two hundreds and eithty-five cases of children hospitalized at Children′s Hospital of Soochow University with MPP(positive for nucleic acid testing of respiratory secretion)were chosen from January 1st, 2019 to September 31st, 2019.Delayed antibody production group included 36 cases, who were tested for negative IgM antibody meanwhile the titer of IgG antibody changed less than 4 folds within 14 days.Positive group included 249 cases who were tested for positive IgM antibody or the titer of IgG antibody changed over 4 folds within 14 days.The characteristics of clinical manifestation, immunology and radiology were comparatively analyzed.Results:The medium age of delayed antibody production group was 0.75(0.30, 2.78)years old, which was obviously younger than that from positive group[5.50(3.73, 7.20)years old]( P<0.001). Low level of serum immunoglobulin IgG was the independent effect factor of delayed production for Mycoplasma pneumoniae antibody( P=0.037). When the serum immunoglobulin IgG level was lower than 7.155mmol/L, the sensitivity of predicting delayed production for mycoplasma pneumoniae antibody would be 0.819 and the specificity was 0.833.The underlying diseases associated with delayed antibody production were hospitalization history during neonatal period( P=0.007)and congenital heart disease( P=0.001). There were 11.11%(4/36)of children appearing spasmodic cough, 41.67%(15/36)of children showing wheezing and 33.33%(12/36)showing diarrhea in delayed antibody group, which were significantly higher than those in positive group[0.40%(1/249), 24.50%(61/249)and 9.64%(24/249), respectively, P<0.05]. The incidence of fever in delayed antibody group were 63.89%(23/36), which was lower than that in positive group[92.37%(230/249)]( P<0.001), meanwhile, the fever last time was 2.50(0, 4.75)days in delayed antibody group, which was shorter than that in positive group[ 7(5.00, 8.50)days]( P<0.001). In the delayed antibody group, there was 19.44%(7/36)of children sufferring from lobar pneumonia, and no extrapulmonary manifestations occurred, which were significantly lower than those in positive group[75.50%(188/249), 14.86%(37/249)]( P<0.05). Conclusion:Delayed antibody production in children with MPP is more common when serum immunoglobulin IgG level is lower than 7.155 mmol/L, especially in the presence of neonatal hospital history and congenital heart disease.The clinical manifestations of these children are mainly characterized by spasmodic cough and wheezing, with low probability of fever, lobular pneumonia and extrapulmonary manifestations.

CRISPR/Cas9 has been widely used in engineering Saccharomyces cerevisiae for gene insertion, replacement and deletion due to its simplicity and high efficiency. The selectable markers of CRISPR/Cas9 systems are particularly useful for genome editing and Cas9-plasmids removing in yeast. In our previous research, GAL80 gene has been deleted by the plasmid pML104-mediated CRISPR/Cas9 system in an engineered yeast, in order to eliminate the requirement of galactose supplementation for induction. The maximum artemisinic acid production by engineered S. cerevisiae 1211-2 (740 mg/L) was comparable to that of the parental strain 1211 without galactose induction. Unfortunately, S. cerevisiae 1211-2 was inefficient in the utilization of the carbon source ethanol in the subsequent 50 L pilot fermentation experiment. The artemisinic acid yield in the engineered S. cerevisiae 1211-2 was only 20%-25% compared with that of S. cerevisiae 1211. The mutation of the selection marker URA3 was supposed to affect the growth and artemisinic acid production. A ura3 mutant was successfully restored by a recombinant plasmid pML104-KanMx4-u along with a 90 bp donor DNA, resulting in S. cerevisiae 1211-3. This mutant could grow normally in a fed-batch fermentor with mixed glucose and ethanol feeding, and the final artemisinic acid yield (> 20 g/L) was comparable to that of the parental strain S. cerevisiae 1211. In this study, an engineered yeast strain producing artemisinic acid without galactose induction was obtained. More importantly, it was the first report showing that the auxotrophic marker URA3 significantly affected artemisinic acid production in a pilot-scale fermentation with ethanol feeding, which provides a reference for the production of other natural products in yeast chassis.
Artemisinins ; Fermentation ; Saccharomyces cerevisiae/metabolism* ; Saccharomyces cerevisiae Proteins/metabolism*

Objective: To investigate the effects of Lactobacillus paracasei N1115 combined with fructooligosaccharides (FOS) on non-alcoholic fatty liver disease (NAFLD) in mice and its possible mechanism. Methods: A total of 50 male C57 mice were randomly and equally divided into five experimental groups. Group 1 received a normal diet (ND). Other four groups received a high-fat diet (HFD) to establish NAFLD models. In addition to HFD, group 3 received Lactobacillus paracasei N1115 (2.2×10⁹ CFU/mL), group 4 received FOS (4 g/kg per day), and group 5 received Lactobacillus paracasei N1115 (2.2×10⁹ CFU/mL) and FOS (4 g/kg per day). All groups received continuous intervention for 16 weeks. The following indices were measured for all groups after intervention: general condition, the levels of fasting blood glucose, insulin, and lipopolysaccharide (LPS), and the levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and interferon (IFN)-γ in the serum and liver. The mRNA levels of Toll-like receptor (TLR)4, nuclear factor (NF)-κb, insulin receptor (InsR), and insulin receptor substrate (IRS)-1 were measured by real-time RT-PCR. The data were subjected to one-way analysis of variance and comparison between groups was made by Bonferroni method. Results: Compared with group 2, groups 3, 4, and 5 had significantly lower body weight, Lee's index, liver index, and the levels of blood glucose and insulin resistance (P < 0.05). The serum level of LPS in group 2 was significantly higher than that in the other experimental groups (group 1: 8.80 ± 0.85 U/L, group 3: 12.31 ± 1.01 U/L, group 4: 12.27 ± 0.98 U/L, and group 5: 10.17 ± 0.79 U/L vs group 2: 15.45 ± 1.14 U/L, F = 55.117, P < 0.001). The levels of TNF-α, IL-1β, IL-6, and IFN-γ in the serum and liver in group 2 were also significantly higher than those in the other groups (P < 0.05). Group 2 had significantly higher mRNA levels of TLR4 and NF-κb in the liver than the other groups (F = 82.933, P < 0.001; F = 149.033, P < 0.001); however, it had significantly lower mRNA levels of InsR and IRS-1 in the liver than the other groups (F = 33.347, P < 0.001; F = 70.225, P < 0.001). Conclusion Lactobacillus paracasei N1115 combined with FOS can reduce the level of LPS in the blood circulation, inhibit activation of the LPS/TLR4 signaling pathway, and reduce the release of inflammatory factor and the body's insulin resistance, so it can relieve NAFLD.

It is one of key scientific problems to identify the effective compounds in the study on new TCM. Because of the integrity of the effects of TCM, the diversity of constituents, the complexity of the targets and the unpredictability of the interaction among the components, the efficiency of the traditional efficacy substance research method is generally not high. Based on the research thought of quantitative composition-activity relationship, there are inevitable quantitative correlation between chemical compositions and its biological activity. The quantitative composition-activity relationship research is an effective method to detect the effective compounds. This article reviewed the common methods and technologies used in the quantitative composition-activity relationship research, with a purpose to provide some references for the study on basic effective components in TCM.

Objective To investigate the effectiveness of Lactobacillus paracasei N1115 combined with fructooligosaccharides (FOS) in the treatment of nonalcoholic fatty liver disease(NAFLD) in a mouse model and to analyze the possible mechanism.Methods Fifty male C57BL/6 mice were randomly divided into five groups and respectively given normal diet (ND group), high-fat diet (HFD group), HFD containing Lactobacillus paracasei N1115 (HFD+L) (2.2×109 CFU/ml), HFD containing FOS (HFD+FOS) (4 g/kg per day) and HFD containing Lactobacillus paracasei N1115 and FOS for 16 consecutive weeks.Levels of triglyceride (TG), total cholesterol (TC), low-density lipoprotein (LDL), high-density lipoprotein (HDL), lipopolysaccharide (LPS) and diamine oxidase (DAO) in serum samples from each group were measured.Expression of tight-junction proteins (Claudin-1 and Occludin), p38 and phosphorylated p38 (p-p38) in intestinal tissues were analyzed.Results Compared with the HFD group, the HFD+FOS+L group showed decreased levels of TC, TG, LDL, LPS and DAO in serum samples, but increased serum HDL level (P<0.05).Moreover, combined treatment with Lactobacillus paracasei N1115 and FOS alleviated liver lipid deposition, significantly increased the expression of Claudin-1 and Occludin in intestine and inhibited the phosphorylation of p38 (P<0.05).Conclusion Lactobacillus paracasei N1115 combined with FOS may increase the expression of Claudin-1 and Occludin through inhibiting the phosphorylation of intestinal p38, which is conducive to maintaining the intestinal mucosal barrier integrity and alleviating NAFLD.