Objective To perform mutation analysis of survival motor neuron gene 1 (SMN1 in two spinal muscular atrophy (SMA) patients and their parents to evaluate the effects of the two SMN1 gene mutations on the transcript levels of the gene and preliminarily predict their effects on the structure and function of SMN protein.Methods Mutation analysis of SMN1 gene was carried out by multiplex ligationdependent probe amplification,reverse transcript-polymerase chain reaction (RT-PCR) and cloning sequencing.Transmission of the mutations was confirmed by the mutation analysis in patients' parents.The full-length SMN1 (SMN1-fl) transcript levels of the patients carrying these subtle mutations were detected using quantitative RT-PCR.Results The two patients were diagnosed as SMA Ⅱ and SMA Ⅲ.They carried p.Val19GlyfsX21 and p.Ala2Gly SMN1 mutations in SMN1 gene,respectively.Both of the two mutations were originated from their fathers.Compared with the healthy individuals (23.5 ± 4.9),the two patients had a significant reduction in the level of SMN1-fl transcripts (t =3.322,P =0.011 (p.Ala2Gly) ;t =6.964,P =0.000 (p.Val19GlyfsX21)).However,compared with the healthy carriers (14.1 ±4.5),the patient with p.Ala2Gly mutation had no significant reduction in the level of SMN1-fl transcripts (13.9 ±3.6,t =0.058,P =0.955) ; however,the patient with p.Val19GlyfsX21 mutation had a significant reduction (4.9± 2.4,t =3.725,P =0.004).Conclusions Two SMN1 gene mutations are identified in our study.The mutation p.Val19GlyfsX21 is a novel mutation and p.Ala2Gly is firstly reported in Chinese SMA patients.p.Val19GlyfsX21 may possibly lead to decreased SMN1-fl mRNA by nonsense-mediated messenger RNA decay,however,p.Ala2Gly has no obvious effects on the amount of the SMN1-fl transcripts,indicating that its deleterious effect may be occurring at SMN protein level or the function of SMN protein.

BACKGROUND: Classic isolation method of bone marrow mesenchymal stem cells (BMSCs) is Percoll density gradient centrifugation. Blood cell component was removed. However, this method is complicated. Preparation density was needed when isolating dog bone marrow. Moreover, centrifugation was frequent, which had a great damage to cells. OBJECTIVE: To establish methods of the isolation, proliferation, culture and osteoinduction of canine BMSCs, and observe the in vitro proliferation and ability to osteoinduction differentiation. METHODS: 10 mL bone marrow was extracted from dog posterior superior iliac spine, heparin anticoagulation, diluted using Hanks juice, treated with 1.077 g/mL Ficoll solution 3 mL, and centrifuged at 2 000 r/min for 20 minutes. Karyocytes were absorbed to form white cloudlike layering interface, and then centrifuged twice using DMEM supplemented with fetal bovine serum, incubated at 12×10~4/cm~2 at 37 ℃ in a 5% CO_2 incubator. Following subculture, cells were incubated in DMEM containing dexamethasone, β-sodium phosphoglycerol and ascorbic acid 2-phosphate. Immunocytochemical staining and immunofluorescence staining were utilized to detect osteocalcin, osteopontin and type Ⅰ collagen expression in osteoblasts. Alkaline phosphatase staining and alizarin red staining were performed. RESULTS AND CONCLUSION: 1.077 g/mL Ficoll density gradient centrifugation was used to isolate karyocytes that were significant compared with Percoll solution. Obtained BMSCs had high purity, good growth and the mean doubling time was 24 hours. Following in vitro osteogenic incubation of dog BMSCs, osteocalcin, osteopontin and type Ⅰ collagen showed positive expression. Alkaline phosphatase staining demonstrated bluish-green cytoplasm. Alizarin red staining showed red nodes in extracellular matrix, and could differentiate into osteoblasts in vitro.

Objective To analyse the distribution characteristics of major allergens initiating allergic diseases in children from rural Shanghai. Methods Eight hundred children with allergic diseases from rural Shanghai (rural ease group), 450 children with allergic diseases from urban Shanghai (urban ease group) and 100 healthy children from rural Shanghai (rural normal control group) underwent skin prick tests (SPT), and children of rural case group were subdivided into infant group, preschool age group and school age group according to age. The positive rates of allergens and SPT were compared among groups. Results The positive rate of SPT of rural case group was significantly higher than that of rural normal control group (73.38% vs 26.00%, P<0.05), and was significantly lower than that of urban ease group (73.38% vs 80.22%, P<0.05). Dermatophagoidesfarinae and Dermatophagoides pteronyssinus were the major allergens in rural ease group, with the positive rates of 57.88% and 59.13%, respectively. Except weed and rubber, there were significant differences in positive rates of the other allergens between rural ease group and the other two groups(P<0.05). There were significant differences in positive rates of SPT among different age groups of rural children with allergic diseases (P<0.05). Conclusion Dermatophagoides farinae and Dermatophagoides pteronyssinus are the major allergens in children with allergic diseases from rural Shanghai, whose positive rates of SPT are lower than those of children with allergic diseases from urban Shanghai. The positive rate of SPT is related to age to some extent.

OBJECTIVETo study the effects of Ligusticum and its two components (Sodium Ferulate and Ligustrazine as main efficacy components in Ligusticum for invigorating blood circulation) on muscle atrophy in a hind limb unloaded rat model.

METHODSThe tail-suspended rats were subjected to a 14-days disuse, immunohistochemistry and hemorheology were used to study the effects of medicines on soleus muscle.

RESULTSCompared with HLU+ W: (1) The CSA of soleus type I fibers in HLU + SfH and HLU+ TmpH increased by 37.3% and 39.4% respectively (P < 0.05). (2) Expression level of MHC II were inhibited in all treatment groups (P < 0.01). (3) Expression of MHC II in nuclear bag 2 fiber were altered from positive to negative. (4) The blood viscosity in low shear rates decreased obviously (P < 0.01), even near to control.

CONCLUSIONLigusticum and its two main efficacy components (Sodium Ferulate and Ligustrazine) can prevent soleus atrophy induced by disuse, and Sodium Ferulate and Ligustrazine in high dose showed most efficacy.

Animals ; Coumaric Acids ; pharmacology ; Drugs, Chinese Herbal ; pharmacology ; Extremities ; Female ; Hemorheology ; Hindlimb Suspension ; Ligusticum ; chemistry ; Muscle Fibers, Skeletal ; drug effects ; Muscular Atrophy ; blood ; etiology ; prevention & control ; Myosin Heavy Chains ; metabolism ; Pyrazines ; pharmacology ; Random Allocation ; Rats ; Rats, Sprague-Dawley

Objective To analyze the distribution of common chromosomal karyotypes of patients with Turner syndrome (TS), and to explore the correlation between the age and height standard deviation scores (HSDS) on diagnosis.Methods Retrospective investigation was performed for the data of age and HSDS on diagnosis in 273 TS girls(≤ 18.0 years old)diagnosed by chromosomal karyotypes.The main statistical methods were analyzed with t-test and Pearson correlation test by using the SPSS 18.0 statistical software.Results (1) There were 4 kinds of common chromosomal karyotypes in the TS :45, X (87/273 cases,31.9％),46, X, i (Xq) (43/273 cases, 15.7％) ,45, X/46, X, i (Xq) (36/273 cases, 13.2％) and 45, X/46, XX (23/273 cases, 8.4％), respectively, the adolescent TS all had delayed puberty.For the cases with 45, X karyotypes ,3 cases presented mental retardation and 2 cases with organs deformity.(2)The patients with 45 ,X/46,X,i(Xq) karyotypes or with 46,X,i(Xq) karyotypes had the maximum(12.56 age) or the minimum(9.70 age) mean age on diagnosis, respectively, there was a significant difference between 2 groups (t =3.019, P =0.004).The maximum deviation from normal height was found in the patients with karyotypes of 46, X,i (Xq) (HSDS =-4.04), and the minimum deviation was in the patients with karyotypes of 45,X/46, XX (HSDS =-3.16), and there was a significant difference between 2 groups (t =-2.95, P =0.004).(3) More than 75.7％ of TS patients was diagnosed when their heights deviated above 3 SD,and their mean age on diagnosis was 12.10 age,which was 3 years later than those patients within 2 SD.(4) There was a significant negative correlation between the age and HSDS on diagnosis in the groups of common chromosomal karyotypes[45,X、46,X,i(Xq) and 45,X/46,XX] (r =-0.551,-0.560,-0.622,all P ＜ 0.01), except for the group with the 45, X/46, X, i (Xq).Conclusions (1) In this study, the consti-tuent ratios of these 4 common chromosomal karyotypes were different from those in Europe and America's.(2)Patients with 45 ,X may have more severe symptoms than others.(3)The mean age on diagnosis was at least 3.0 years earlier when considered HSDS below-2.00 as an indicator for chromosomal karyotype screening,which would facilitate earlier diagnosis.

BACKGROUND: Inhibiting the degradation of extracellular matrix in the intervertebral disc can delay the degenerative process of intervertebral disc. Matrix metalloproteinases-3 (MMP3) is considered as a key enzyme for degradation of extracelular matrix components such as type II collagen and aggrecan.
OBJECTIVE:To construct the short hairpin RNA lentiviral vector targeting human MMP3 gene and to detect its efficiency of gene silence by infecting human degenerative nucleus pulposus cells.
METHODS:According to the human MMP3 mRNA (NM_002422.4) sequence, four groups of the short hairpin RNA gene sequences targeting MMP3 were designed, synthesized and annealed to form double stranded DNA fragments, which were connected with the LV3 vectors digested by BamHI andEcoRI enzymes, and then transfected into the competent cels. The positive clones were identified by PCR, and analyzed by sequencing. The packaging and titer of lentivirus were determined after transfecting 293T cells. Human degenerative nucleus pulposus cels were infected with lentivirus vector, and the transfection efficiency of each group was observed under inverted fluorescence microscope. The interfering efficiency was detected by real time-PCR and western blot at 72 and 96 hours.
RESULTS AND CONCLUSION:The ds-oligo DNA was successfully inserted into the lentiviral vector as confirmed by electrophoresis and sequence analysis. The recombinant lentivirus was harvested from 293T cels with a viral titer of 1-5 ×108 TU/mL. RNA interference targeting the GCC AGG CTT TCC CAA GCA AAT sequences with the highest interfering efficiency in MMP3 gene at 72 and 96 hours resulted in suppression of MMP3 mRNA expression by 98% and 72%, respectively; and at 96 hours, the interfering efficiency of protein expression was 57.2%. The recombinant lentivirus vector containing RNA interference targeting MMP3 gene is successfuly constructed, which lays a foundation for further studies on the MMP3 function and gene therapy.

Adolescent ; Cardiac Catheterization ; methods ; Child, Preschool ; Echocardiography, Transesophageal ; methods ; Female ; Heart Septal Defects, Atrial ; diagnostic imaging ; therapy ; Humans ; Treatment Outcome

This paper was aim to screen microorganisms with attenualed efficiency for Chinese medicine containing aristolochic acid A by liquid-state fermentation. Twelve Chinese medicine were detected by UPLC and aristolochic acid A was only founded in four species of Aristolochia, those were Caulis Aristolochiae Manshuriensis, Aristolochiae Radix, Aistolochia Contorta Bunge and Herba Aristolochiae Mollissima,but not in the others. With the four Chinese medicine containing aristolochic acid A as raw material, ten microorganisms were tested, and the content of aristolochic acid A was detected by UPLC. The results showed that one microorganism can decrease content of aristolochic acid A in all those four Chinese medicine.
Aristolochic Acids ; analysis ; metabolism ; Bacteria ; metabolism ; Biotransformation ; Drugs, Chinese Herbal ; analysis ; metabolism ; Fungi ; metabolism ; Plants, Medicinal ; chemistry ; microbiology

AIM: To investigate the changes and effects of orthokeratology on corneal morphology. METHODS: Totally 90 patients were treated with orthokeratology from January 2015 to December 2016. They were divided into observation group (overnight wearing) and control group(daytime wearing),45 cases (90 eyes) in each. The central corneal thickness, corneal curvature, spherical equivalent (SE), uncorrected visual acuity (UCVA) of both groups were compared before and after wearing orthokeratology lens for 1wk,1,3,and 6mo. RESULTS: The central corneal thickness of two groups before wearing glasses was significantly higher than that of the cornea after wearing glasses 1wk,1,3 and 6mo(all P<0.05); the central corneal thickness of the observation group at 1, 3 and 6mo after wearing glasses was significantly lower than that of the control group (P<0 05). The corneal curvature values of the two groups before wearing glasses were significantly higher than that of the cornea after wearing for 1wk,1,3 and 6mo (all P<0.05). The corneal curvature of observation group at 6mo was significantly lower than that of the control group (40.0士0.5D vs 41.3士0.9D, P<0.05). The staining rate of corneal epithelium was observed at 3mo after wearing glasses. The positive rate of epithelial staining was 49% (44/90) in the observation group and 29% (26/90) in the comparison group;the grade 0,grade 1 accounted for the majority of the two groups. With the orthokeratology lens wearing longer, the SE level of two groups showed a downward trend. The spherical equivalent of observation group at 6mo was significantly lower than that of the control group (-0.42士0.20D vs -0 52士0.19D, P<0.05). The UCVA value of two groups after wearing glasses significantly increased than that before wearing glasses (all P<0.05). CONCLUSION: Wearing orthokeratology lens can reduce myopia degree. Wearing it overnight has the better outcome than wearing in the daytime.

BACKGROUNDMutations in fumarylacetoacetate hydrolase (FAH) gene can lead to tyrosinemia type 1 (HT1), a relatively rare autosomal recessive disorder. To date, no molecular genetic defects of HT1 in China have been described. We investigated a Chinese family with a HT1 child to identify mutations in FAH.

METHODSDNA sequencing was used for mutations screening in FAH gene. Real-time polymerase chain reaction (PCR) was performed to determine the FAH gene expression level. To confirm the presence of degradation by the nonsense-mediated mRNA decay pathway (NMD), the fragments containing R237X mutations were analyzed by primer introduced restriction analysis-polymerase chain reaction (PIRA-PCR) and cDNA sequencing. Finally, the effects of the mutations reported in this study were predicted by online softwares.

RESULTSA boy aged 3 years and 8 months was diagnosed clinically with HT1 based on his manifestations and biochemical abnormalities. Screening of FAH gene revealed two heterozygous mutations R237X and L375P transmitted from his mother and father respectively. In this pedigree, the amount of FAH mRNA relative to a healthy control was 0.44 for the patient, 0.77 for his mother and 1.07 for his father. Moreover, both PIRA-PCR and cDNA sequencing showed significant reduction of the FAH mRNA with R237X nonsense mutation. The missense mutation of L375P was not reported previously and prediction software showed that this mutation decreased the stability of protein structure and affected protein function.

CONCLUSIONSThis is the first case of HT1 analyzed by molecular genetics in China. The R237X mutation in FAH down- regulates the FAH gene expression, and the L375P mutation perhaps interrupts the secondary structure of FAH protein.

Child, Preschool ; China ; Humans ; Hydrolases ; genetics ; Male ; Molecular Sequence Data ; Mutation ; Mutation, Missense ; genetics ; Nonsense Mediated mRNA Decay ; genetics ; Real-Time Polymerase Chain Reaction ; Tyrosinemias ; genetics