Objective To construct recombinant adenovirus vector containing mouse CD40Ig fusion gene for the study of induction of donor-specific tolerance. Methods CD40Ig fusion gene was constructed by PCR overlapping technique, and was cloned into the shuttle plasmid pAdTtrack-CMV. The linearized shuttle plasmid was co-transfected into the E.coli strain BJ5183 with bone plasmid pAdeasy1, then the recombinant adenovirus plasmid was generated. The adenovirus was packaged and amplified in Cells 293. Results The recombinant virus AdCD40Ig was successfully constructed and its titer was 5?109 efu/ml. Conclusion AdCD40Ig can be used as an agent in experiment to induce donor-specific tolerance.

OBJECTIVE:To identify a new adulterated dye,and to establish simultaneous detection method for 9 orange-yel-low dyes in Typhae Pollen. METHODS:TLC and LC-MS were used for the identification of the new adulterated dye;TLC and HPLC method were used for determining 9 orange-yellow dyes. The determination was performed on Welch AQ-C18 column with mobile phase consisting of acetonitrile-0.05 mol/L ammonium acetate solution (gradient elution) at the flow rate of 1.0 mL/min. The detection wavelength were set at 432 nm (lemon yellow,metanil yellow,gold orange O,basic orangeⅡ) and 484 nm (sun-set yellow,acid orange Ⅰ,gold orangeⅡ,orange G,basic orange 21). The column temperature was 35 ℃.The sample size was 10 μL. RESULTS:TLC spots of auramine O,lemon yellow and new adulterated dye were clear and well separated without in-terference from negative control;new adulterated dye was identified as metanil yellow. TLC spots of lemon yellow,sunset yellow, orange G,acid orangeⅠ,gold orangeⅡ,auramine O,metanil yellow,basic orange 21 and basic orangeⅡwere clear and well sep-arated without interference from negative control. The limits of detection were 0.30,0.20,0.33,0.16,0.19,0.19,0.31,0.26, 0.30 mg/kg. CONCLUSIONS:The established method is able to detect adulterated dye and orange-yellow dye in Typhae Pollen rap-idly and accurately.

Objective:To explore the effect of low-frequency repetitive transcranial magnetic stimulation (rTMS) on the executive functioning and core symptoms of preschool children on the autism spectrum.Methods:Forty-three preschool children showing signs of autism were randomly divided into an rTMS group of 21 and a control group of 22. In addition to routine rehabilitation and training in basic living skills, the rTMS group was additionally provided with 1Hz rTMS for 8 weeks. Before and after the treatment, both groups were evaluated using the preschool version of the Behavior Rating Inventory for Executive Function (BRIEF-P), the Social Responsiveness Scale (SRS), the revised version of the Repetitive Behavior Scale (RBS-R) and the Child Autism Rating Scale (CARS).Results:After the 8 weeks of treatment, the average BRIEF-P, SRS, RBS-R and CARS scores of both groups had improved significantly, but the rTMS group′s averages where then significantly better than those of the control group.Conclusions:Low-frequency rTMS in addition to conventional rehabilitation intervention can significantly improve the executive functioning and core symptoms of preschool children on the autism spectrum.

Objective:To observe any effect of repetitive low-frequency transcranial magnetic stimulation (rTMS) on sleep disorders and abnormal behaviors of children on the autism spectrum.Methods:Forty autistic children were randomly divided into an observation group and a control group, each of 20. Both groups were given sleep behavior training and individualized conventional rehabilitation training. Those in the observation group also received 30min of rTMS at 1Hz applied over the left dorsolateral prefrontal cortical area once a day, 5 days a week. Before and after 8 weeks of this treatment, both groups were evaluated using the Autism Behavior Checklist (ABC), the Child Autism Rating Scale (CARS) and the children′s Sleep Habit Questionnaire (CSHQ).Results:The average CARS and CSHQ scores, as well as the total ABC score of both groups increased significantly over the 8 weeks, but the average CARS and CSHQ scores, as well as the total ABC score of the observation group were then significantly better than in the control group. After the treatment, the average ABC scores for sensory ability, communication ability, motor ability, and language ability were significantly lower than before the treatment for both groups, but the observation group′s averages were then significantly better than those of the control group.Conclusions:Supplementing routine intervention with low-frequency rTMS can effectively improve the sleeping and correct the abnormal behavior patterns of autistic children.

OBJECTIVESTo evaluate the expression profile of myoD microRNA-29 (miR-29) family in L6 myoblast differentiated to myotube of L6 myotube treated by glucose and insulin, and to further probe the molecular mechanism of myoD regulating the expression of miR-29 clusters.

METHODSThe expression of myoD and miR-29 family was detected by using real-time PCR and Western blot analysis. The potential promoter and transcription factors binding sites of miR-29 clusters were predicted by Promoter scan and transcriptional factor search. The promoter sequence of miR-29b1-a and miR-29b2-c cluster was cloned into a luciferase reporter plasmid and the regulatory effect of myoD was analyzed by using dual luciferase reporter assay. Electrophoretic mobility shift assay was further conducted to indicate the binding of myoD on specific sequence. Moreover, overexpression of myoD was achieved by a recombinant adenovirus system (Ad-myoD). L6 cells were infected with Ad-myoD and real-time PCR was conducted to analyze the expression of miR-29b and miR-29c.

RESULTSThe expression levels of myoD, miR-29a, miR-29b, and miR-29c were increased in L6 myoblast differentiated to myotube. The expression of myoD, miR-29b, and miR-29c was up-regulated in L6 myotube treated with glucose and insulin, but miR-29a depicted no significant change. Dual luciferase reporter gene assay showed that myoD functioned as a positive regulator of miR-29b2-c expression and myoD could bind to the specific sequence located at the promoter region of miR-29b2-c cluster. Enforced expression of myoD led to a marked increase of miR-29b and miR-29c levels in L6 cells.

CONCLUSIONMyoD might act as a crucial regulator of myogenesis and glucose metabolism in muscle through regulating the expression of miR-29b2-c.

Animals ; Cell Differentiation ; drug effects ; physiology ; Cell Line ; Gene Expression Regulation ; drug effects ; physiology ; Glucose ; pharmacology ; Hypoglycemic Agents ; pharmacology ; Insulin ; pharmacology ; Mice ; MicroRNAs ; biosynthesis ; genetics ; Multigene Family ; physiology ; Muscle Fibers, Skeletal ; cytology ; metabolism ; MyoD Protein ; genetics ; metabolism ; Myoblasts ; cytology ; metabolism ; Sweetening Agents ; pharmacology

OBJECTIVE： To establish a method for measuring the paclobutrazol residue in Ophiopogon japonicus from Sichuan and detect the quality of O. japonicus from Sichuan from different sources. METHODS： Totally 50 batches of samples were collected from different origin places， commercial markets and manufacturers. The sample pretreatment method was QuEChERS method， .ie the sample was extracted by aqueous acetonitrile， salted out by QuEChERS extract package （containing anhydrous magnesium sulfate and anhydrous sodium acetate）， the extract solution was purified by QuEChERS purification package （containing anhydrous magnesium sulfate， N-propyl ethylenediamine， octadecylsilane chemically bonded silica， silica gel， graphitized carbon black） and then added into internal standard triphenyl phosphate. The paclobutrazol residue in O. japonicus from Sichuan was determined by GC-MS/MS. The determination was performed on DB-5MS column. The temperature programming was adopted， and the detector was triple quadrupole MS detector. The initial flow rate of carrier gas was 1.3 mL/min； acquisition mode was MRM. Injection method was splitless injection. RESULTS： The linear range of paclobutrazol was 1.01-505 ng/mL （r＝    0.999 7）. RSDs of precision， stability （24 h） and repeatability tests were 3.94％， 13.62％， 7.54％ （n＝6）， respectively. Average method recovery was 111.26％ （RSD＝5.43％， n＝9）. The paclobutrazol residue in 50 batches of sample were 0.02-2.72 mg/kg. CONCLUSIONS： Established method is simple， accurate， sensitive and reproducible. It also can be used for the determination of paclobutrazol residue in O. japonicus from Sichuan. The contents of paclobutrazol residue in O. japonicus from Sichuan from different sources are different greatly.

Objective : To investigate the effects of D ⁃allose on the restoration of neurological function , Galectin⁃3 (Gal⁃3) , adenosine monophosphate⁃activated protein kinase/mammalian target of rapamycin (AMPK/mTOR) and the expression of some inflammatory factors in ischemia⁃reperfusion injury ( CIRI) mice . Methods : A total of 50 male mice were randomly divided into control group (Con group) , sham group (Sham group) , cerebral ischemia⁃reperfusion injury group (MCAO group) , cerebral ischemia⁃reperfusion injury + D ⁃alolose group (MCAO + D ⁃allose group) and cerebral ischemia⁃reperfusion injury + modified citrus pectin group (MCAO + MCP group) . The middle cerebral artery occlusion/reperfusion (MCAO/R) model (reperfusion after 2 hours of MCA ischemia) was established by thread embolism . After successful modeling , the neurological function of mice was evaluated Longa score and rotated rod walking . TTC staining was used to observe the volume of cerebral infarction foci . The expression levels of Gal⁃3 and autophagy⁃related molecules were detected by Western blot and RT⁃PCR . Immunofluorescence was applied to detect the distribution of Gal⁃3 in brain tissue , and TNF⁃α , IL⁃8 secretion was detected with ELISA KIT . Results : Compared with Con group and Sham group , the MCAO model represented significant increase in the Longa neurofunction score (P < 0. 01) , cerebral infarction volume ( P < 0. 01) , Gal⁃3 expression and manifasted enhanced autophagy (P < 0. 01) . After treatment with D ⁃allose , it could significantly improve neurological dysfunction , reduce cerebral infarction volume (P < 0. 01) , reduce the expression of Gal⁃3 ( P < 0. 01) , inhibit AMPK phosphorylation , promote mTOR phosphorylation , and inhibit autophagy (P < 0. 01) . The use of the Gal⁃3 inhibitor MCP alone could also achieve the effect of inhibiting autophagy . Conclusion D ⁃allose can effectively promote the recovery of neurological function and reduce the volume of infarct foci in CIRI mice . The mechanism may involve inhibiting excessive cell autophagy by downregulating the expression of Gal⁃3 , and reducing the release of inflammatory factors such as TNF⁃α and IL⁃8 , thereby exerting neuroprotective effects .

Objective: To summarize the clinical characteristics of inflammasomopathies, enhance the recognition of those diseases, and help to establish the early diagnosis. Methods: The clinical manifestations including fever, rash, systems involvement as well as laboratory results and genotypic characteristics of 35 children with inflammasomopathies diagnosed by the Department of Pediatrics, Peking Union Medical College Hospital, from January 1, 2008 to December 31, 2020 were analyzed retrospectively. Results: A total of 35 cases of inflammasomopathies were diagnosed, and 20 of them were boys while 15 were girls. Inflammasomopathies patients have early onset, the age of onset as well as diagnostic age were 1 (0,7) and 7 (3,12), respectively. Among those patients, 10 had familial mediterranean fever, 3 had mevalonate kinase deficiency, 15 cases had NLRP3 gene associated autoinflammatory disease, 4 cases had NLRP12-associated autoinflammatory disease, 2 cases had familial cold autoinflammatory syndrome 3, and 1 case had familial cold autoinflammatory syndrome 4. A total of 34 cases (97%) showed recurrent fever, 27 cases (77%) had skin rashes, while 11 cases (31%), 10 cases (29%), and 8 cases (23%) were presented with lymphadenopathy, hepatosplenomegaly and growth retardation, respectively. In terms of systemic involvement, there were 18 cases (51%), 12 cases (34%), 8 cases (23%), and 5 cases (14%) with skeletal, neurological, auditory, and renal involvement, respectively. Central nervous system involvement was seen only in NLRP3 gene associtated autoinflammatory diseases (12 cases), sensorineural deafness was seen in NLRP3 gene associtated autoinflammatory diseases (6 cases) and NLRP12 gene associated autoinflammatory diseases (2 cases), and abdominal pain was observed in familial Mediterranean fever (5 cases), mevalonate kinase deficiency (1 case) and NLRP12 gene related autoinflammatory diseases (1 case). In the acute inflammatory phase, the acute phase reactants (erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP)) of 35 cases (100%) were significantly increased. There were 21 cases received ferritin examination, and only 4 cases (19%) showed an increase of it. In terms of autoantibodies, among all 35 patients, 4 cases (11%) were positive for antinuclear antibodies (ANA). Conclusions: Fever, skin rash, and skeletal manifestations are the most common clinical features, accompanied with increased CRP and ESR, and negative results of autoantibodies such as ANA. The clinical manifestations of those diseases are complex and diverse, and it is prone to delayed diagnosis and treatment.
Child ; Familial Mediterranean Fever ; Female ; Fever/etiology* ; Genotype ; Hereditary Autoinflammatory Diseases ; Humans ; Male ; Retrospective Studies

BackgroundMultiple sclerosis (MS) is a common central nervous system autoimmune disorder. Increasing number of genome-wide association study (GWAS) analyses hint that MS is strongly associated with genetics. Unfortunately, almost all the GWAS analyses were Caucasian population based. Numbers of risk loci might not be replicated in Chinese MS patients. Hence, we performed a MassArray Assay to genotype the previously reported variants located in the transcription regulation genes in order to elucidate their role in the Chinese MS patients.

MethodsOne hundred and forty-two relapsing-remitting MS (RRMS) patients and 301 healthy controls were consecutively collected from September 2, 2008, to June 7, 2013, as stage 1 subjects. Eight reported transcription regulation-related single-nucleotide polymorphisms (SNPs) were genotyped using the Sequenom MassArray system. In stage 2, another 44 RRMS patients and 200 healthy controls were consecutively collected and Sanger sequenced from April 7, 2015, to June 29, 2017, for the validation of positive results in stage 1. Differences in allele and genotype frequencies between patients and healthy controls, odds ratios, and 95% confidence intervals were calculated with the Chi-square test or Fisher's exact test. Hardy-Weinberg equilibrium was tested also using the Chi-square test.

ResultsIn stage 1 analysis, we confirmed only one previously reported risk variant, rs11129295 in EOMES gene. We found that the frequency of T/T genotype was much higher in MS group (χ = 10.251, P = 0.005) and the T allele of rs11129295 increased the risk of MS (χ = 10.022, P = 0.002). In stage 2 and combined analyses, the T allele of rs11129295 still increased the risk of MS (χ = 4.586, P = 0.030 and χ = 16.378, P = 5.19 × 10, respectively).

ConclusionsThis study enhances the knowledge that the variant of EOMES is associated with increasing risk in Chinese RRMS patients and provides a potential therapeutic target in RRMS.

Adolescent ; Adult ; Aged ; Alleles ; Asian Continental Ancestry Group ; Female ; Gene Frequency ; genetics ; Genetic Predisposition to Disease ; genetics ; Genome-Wide Association Study ; Genotype ; Humans ; Male ; Middle Aged ; Multiple Sclerosis ; genetics ; Odds Ratio ; Polymorphism, Single Nucleotide ; genetics ; T-Box Domain Proteins ; genetics ; Young Adult

Objective:To explore the potential synergistic protective mechanism of Glycyrrhizae Radix et Rhizoma-Granati Pericarpium formula compound by using the methods and tools of network pharmacology，and provide a basis for the modernization of traditional Chinese medicine（TCM） compounds and the discovery of new drugs. Method:Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform （TCMSP） was used to obtain the active components of Glycyrrhizae Radix et Rhizoma-Granati Pericarpium formula and their corresponding targets. The obtained targets were input to the UniProt database to inquire the gene names corresponding to the targets. By searching the CTD database，Genecards database and OMIM database of disease-related websites，the anti-sunburn targets were obtained. The interaction of the active targets was analyzed with online STRING database to screen the predicted core targets. The gene ontolog（GO） gene function enrichment analysis and Kyoto encyclopedia of genes and genomes（KEGG） pathway enrichment analysis of the predictive targets were performed by using DAVID database. Cytoscape 3.6.1 software was used to make "drug-component-target" network diagram，"protein-protein interaction" network diagram and "component-target-pathway" network diagram. Online website Draw Venn Diagram was used to show the relationship between disease targets and drug predicted targets. R Studio software was used to draw the functional enrichment analysis diagram of GO gene and KEGG pathway. Molecular docking between the active ingredients and the core targets was performed using GOLD software. Result:The 16 active compounds were collected，such as liquiritin，glycyrrhizin，kaempferol and quercetin. The active components mainly acted on 5 core targets：protein kinase B1（AKT1），interleukin（IL）-6，vascular endothelial growth factor（VEGFA），tumor necrosis factor（TNF） and tumor suppressor gene （TP53） and played a role in anti-sunburn effect primarily through these pathways such as hepatitis B，pathways in cancer，toxoplasmosis，chagas disease（American trypanosomiasis），and TNF signaling pathway. Conclusion:Based on the method of network pharmacology，the present study has preliminarily explored the anti-sunburn targets and pathways of Glycyrrhizae Radix et Rhizoma-Granati Pericarpium formula，and further verified the characteristics of multi-component and multi-target treatment of diseases in TCM，so as to provide certain scientific ideas for the modernization research of Chinese herbal compound prescriptions.