ObjectiveTo investigate the possible mechanism of Buzhong Yulin Decoction （补中愈淋汤） in the prevention and treatment of recurrent urinary tract infection. MethodsThe mouse models of recurrent urinary tract infection were established by uropathogenic Escherichia coli （UPEC） strain UTI89 by bladder perfusion， and the successful mouse models were randomly divided into a model group， an antibiotic group， and a low- and high-dose Buzhong Yulin Decoction group， with six mice in each group. In addition， 5 C57BL/6 mice without modelling were taken as blank group. The low- and high-dose Buzhong Yulin Decoction groups received 0.1 ml/10 g of decoction by gavage， with concentrations of 1.3 g/ml and 5.2 g/ml， respectively； the antibiotic group received 0.1 ml/10 g of levofloxacin hydrochloride solution with 5 mg/ml by gavage； the blank and model groups received 0.1 ml/10 g of distilled water by gavage. Each group was gavaged once a day for 7 consecutive days. The total number of urine marks， the number of central urine marks， and the total urine volume of the urine marks were observed by the urine marking test； HE staining was used to observe the histopathological changes in the bladder of mice； serum levels of the inflammatory factors interleukin 1β （IL-1β）， interleukin 6 （IL-6） and tumour necrosis factor α （TNF-α） were detected by ELISA； the morphology of the epithelial cells of bladder was observed by scanning electron microscopy； immunofluorescence assay to detect bladder tissue anti-UroPlakin 3A protein level and UPEC bacterial load； the spread plate method to detect urinary bacterial load and bacterial load of bladder epithelial cells； RT-PCR method to detect Ras-related protein Rab-11A （RAB11A） and Ras-related protein Rab-27B （RAB27B） mRNA level in bladder tissue； immunoblotting to detect microtubule-associated protein 1 light chain3 （LC3） and P62 protein levels in bladder tissue. ResultsCompared with the blank group， the bladder epithelial cell layers were lost and showed abnormal morphology in mice of the model group； bladder tissue UroPlakin 3A protein and RAB11A and RAB27B mRNA levels reduced， the total number of urine marks， the number of central urine marks， bladder tissue UPEC bacterial load， urinary bacterial load， bacterial load in bladder epithelial cells， serum IL-1β， IL-6， and TNF-α levels， and LC3 and P62 protein levels in bladder tissue all elevated （P＜0.05 or P＜0.01）. Compared with the model group， the bladder epithelial cell layers were intact and the morphology of epithelial cells were regular in the low- and high-dose Buzhong Yulin Decoction groups； the average surface area of bladder epithelial cells reduced， the levels of UroPlakin 3A protein and RAB11A and RAB27B mRNA in bladder tissues elevated， and total number of urine marks， the number of central urine marks， bladder tissue UPEC bacterial load， urinary bacterial load， bacterial load in bladder epithelial cells， serum IL-1β， IL-6， and TNF-α levels， and P62 protein levels in bladder tissue all reduced （P＜0.05 or P＜0.01）， but LC3 protein levels showed no statistically significant （P＞0.05）. In the antibiotic group， the bladder epithelial cells were partially missing and the morphology of epithelial cells was abnormal. Compared with the antibiotic group， the average surface area of the bladder epithelial cells in the mice increased in the low- and high-dose Buzhong Yulin Decoction groups， the bacterial load of the bladder epithelial cells decreased， and the P62 protein level of the bladder tissue decreased （P＜0.05）. When comparing between the low- and high-dose Buzhong Yulin Decoction groups， the differences in each index were not statistically significant （P＞0.05）. ConclusionBuzhong Yulin Decoction may prevent and treat recurrent urinary tract infection by repairing the bladder mucosal barrier， increasing RAB11A and RAB27B level and enhancing autophagy in bladder tissues， thereby facilitating bacterial clearance from bladder epithelial cells and reducing the bacterial load of bladder epithelial cells.

To investigate effects of MARCH6 gene knockdown on MCF-7 cell proliferation and cell cycle.
 Methods: 293T cells were transfected with MARCH6 shRNA lentivirus. Fluorescence microscope was used to observe and verify the transfection efficiency. The initial effect of the MARCH6 gene knockdown in MCF-7 cells was observed via fluorescence microscope. Real-time PCR and Western blot were used to detect the expression of MARCH6. MTT and BrdU assay were used to examine cell proliferation, and staining flow cytometry was used to analyze cycle distribution of MCF-7 cells.
 Results: MARCH6 shRNA lentivirus was successfully transfected and about 80% of the cells expressed green fluorescent in comparison of the control. About 90% of the cells showed green fluorescence. The mRNA and protein in MCF-7 cells were transcription and expression of protein was significantly decreased after the transfection of MARCH6 shRNA lentivirus accompanied by a decrease in MCF-7 cell proliferation (P<0.01). Flow cytometry showed that the cell cycles were inhibited at the G1 phase and the proliferation index was significantly reduced.
 Conclusion: Knockdown of MARCH6 gene by RNA interference inhibits the proliferation of MCF-7 cells, suggesting that the expression of MARCH6 promotes proliferation of breast cancer cells through regulation of the cell cycle.
Adenocarcinoma ; genetics ; Breast Neoplasms ; genetics ; Cell Cycle ; Cell Division ; Cell Proliferation ; genetics ; Female ; G1 Phase ; genetics ; Gene Knockdown Techniques ; Humans ; Hyperplasia ; Lentivirus ; MCF-7 Cells ; physiology ; Membrane Proteins ; physiology ; RNA Interference ; RNA, Messenger ; RNA, Small Interfering ; Real-Time Polymerase Chain Reaction ; Transfection ; Ubiquitin-Protein Ligases ; physiology

OBJECTIVETo study the changes in quantitative kinetic parameters in dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) during radiotherapy and their value for efficacy evaluation in patients with nasopharyngeal carcinoma (NPC).

METHODSTwenty-four patients with NPC that had been pathologically confirmed as poorly differentiated squamous cell carcinoma underwent conventional MRI and DCE-MRI scans 1-2 days before radiotherapy (Pre-RT), during radiotherapy (RT 50 Gy), and upon completion of radiotherapy (RT 70 Gy). Based on the two-compartment model and using the arterial input function deconvolution technique, we calculated the quantitative kinetic parameters of DCE-MRI (K(trans), kep, and Ve) of the tumor tissues, examined the correlation between the tumor regression rate (RS0-50) and the parameters on Pre-RT and RT 50 Gy, and compared the parameters for RT 70 Gy among the groups with different prognosis.

RESULTSThe K(trans) value of the tumor tissue decreased after radiotherapy and showed a significant difference between Pre-RT and RT 70 Gy, but not between Pre-RT and RT 50 Gy. The kep value decreased and Ve value increased after radiotherapy. The tumor regression rate was found to be positively correlated with the K(trans) value for Pre-RT (P=0.005) but negatively with the K(trans) value for RT 50 Gy (P=0.001). During the follow-up for 3 years, 5 patients died and 3 patients had distant metastases. No statistical differences in K(trans), kep, or Ve were found between the groups with different prognosis.

CONCLUSIONSThe kinetic parameters in DCE-MRI, which vary significantly during radiotherapy, allow monitoring of tumor angiogenesis and vascular permeability and quantitative assessment of treatment efficacy for NPC. K(trans) value for Pre-RT and RT 50 Gy can serve as an indicator for early efficacy assessment of radiotherapy and for treatment adjustment, but its relation with the long-term outcomes awaits further study.

Algorithms ; Capillary Permeability ; Carcinoma ; Carcinoma, Squamous Cell ; diagnosis ; Humans ; Magnetic Resonance Imaging ; Nasopharyngeal Neoplasms ; blood supply ; radiotherapy ; Neovascularization, Pathologic ; Prognosis ; Treatment Outcome

Objective To investigate radio-sensitivity and expression of GRP78 protein in the survival subclones of nasopharyngeal carcinoma (NPC) C666-1 cells.Methods NPC C666-1 cells were first irradiated with X-ray at a large dose of 8Gy.Three survival subclones were selected and named as C666-1-R1, C666-1-R2, and C666-1-R3.The radio-sensitivity was analyzed for the radiated survival subclones and their parent control C666-1 cells with Methyl Thiazolyl Tetrazolium assay(MTT) and Trypan blue dye methods.The expression of GRP78 was analyzed for three survival subclones and control C666-1 with Western blot.Results After 6 Gy irradiation, the cell survival rate of three subclones was higher than that of the control cells, especially a significant difference for C666-1-R2 cells (P ＜ 0.05), which suggested a radioresistance in C666-1-R2 cells.Moreover, GRP78 expression in each subclone was significantly higher than that of parent C666-1 cells (P ＜ 0.05).Conclusions The irradiated-survival subclone C666-1-R2 was radio-resistant.GRP78 was overexpressed in the irradiated-survival subclones.GRP78 might be an ideal target for treatment of a nasopharyngeal carcinoma.

Objective To characterize the mutation of phenylalanine hydroxylase (PAH) gene in patients with phenylketonuria(PKU) in Ningxia area,China.Methods Seventy-three children diagnosed with PKU at the Child and Maternal Healthcare Hospital of Ningxia Hui Autonomous Region between January 2010 and June 2013,and 100 non-PKU children randomly chosen from children with normal results in PKU screening were enrolled in the study.Venous blood was collected and the PAH gene sequence was determined by direct DNA sequencing after amplification with the polymerase chain reaction technique.The new gene mutations were defined based on the national and international literature search and databases.The source of the newly discovered mutations was also measured by examining and sequencing the blood samples of their parents.The Chi-square test was used for statistical analysis.Results Among 146 alleles of the 73 PKU children,the detection rate of mutation of PAH gene was 79.5％ (116/146),including 37 types of mutations occurring in 11 exons other than exon 2 and exon 13.The 37 different mutations included 22 missense mutations (59.5％,22/37),six nonsense mutations(16.2％,6/37),six splice site mutations(16.2％,6/37) and three deletion mutations(8.1％,3/37).p.R243Q(17.1％,25/146),EX6-96A ＞ G (6.8％,10/146),p.R241C(6.2％,9/146),p.R413P (5.5％,8/146),p.Rl11X(4.8％,7/146) and IVS4-1G ＞ A(4.8％,7/146) were found to have a higher mutation frequency.Meanwhile,p.R243Q was the most common mutation among Han and Hui ethnic groups with a frequency of 18.8％(12/64) and 15.9％ (13/82),respectively.In contrast,p.R241C showed a significant higher frequency in the Hui group [9.8％(8/82) vs 1.6％(1/64),x2=4.17,P=0.04].Four new mutations of PAH genes,including p.Q304K,p.H107R,p.F392I and p.N223I,were discovered after literature search and comparative studies.Conclusions PAH gene mutations in children with PKU in Ningxia area are unique and are characterized by the diversity and complexity of mutation occurrence in this ethnic region.

Objective To study the changes in quantitative kinetic parameters in dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) during radiotherapy and their value for efficacy evaluation in patients with nasopharyngeal carcinoma (NPC). Methods Twenty-four patients with NPC that had been pathologically confirmed as poorly differentiated squamous cell carcinoma underwent conventional MRI and DCE-MRI scans 1-2 days before radiotherapy (Pre-RT), during radiotherapy (RT 50 Gy), and upon completion of radiotherapy (RT 70 Gy). Based on the two-compartment model and using the arterial input function deconvolution technique, we calculated the quantitative kinetic parameters of DCE-MRI (Ktrans, kep, and Ve) of the tumor tissues, examined the correlation between the tumor regression rate (RS0-50) and the parameters on Pre-RT and RT 50 Gy, and compared the parameters for RT 70 Gy among the groups with different prognosis. Results The Ktrans value of the tumor tissue decreased after radiotherapy and showed a significant difference between Pre-RT and RT 70 Gy, but not between Pre-RT and RT 50 Gy. The kep value decreased and Ve value increased after radiotherapy. The tumor regression rate was found to be positively correlated with the Ktrans value for Pre-RT (P=0.005) but negatively with the Ktrans value for RT 50 Gy (P=0.001). During the follow-up for 3 years, 5 patients died and 3 patients had distant metastases. No statistical differences in Ktrans, kep, or Ve were found between the groups with different prognosis. Conclusions The kinetic parameters in DCE-MRI, which vary significantly during radiotherapy, allow monitoring of tumor angiogenesis and vascular permeability and quantitative assessment of treatment efficacy for NPC. Ktrans value for Pre-RT and RT 50 Gy can serve as an indicator for early efficacy assessment of radiotherapy and for treatment adjustment, but its relation with the long-term outcomes awaits further study.

Objective To study the changes in quantitative kinetic parameters in dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) during radiotherapy and their value for efficacy evaluation in patients with nasopharyngeal carcinoma (NPC). Methods Twenty-four patients with NPC that had been pathologically confirmed as poorly differentiated squamous cell carcinoma underwent conventional MRI and DCE-MRI scans 1-2 days before radiotherapy (Pre-RT), during radiotherapy (RT 50 Gy), and upon completion of radiotherapy (RT 70 Gy). Based on the two-compartment model and using the arterial input function deconvolution technique, we calculated the quantitative kinetic parameters of DCE-MRI (Ktrans, kep, and Ve) of the tumor tissues, examined the correlation between the tumor regression rate (RS0-50) and the parameters on Pre-RT and RT 50 Gy, and compared the parameters for RT 70 Gy among the groups with different prognosis. Results The Ktrans value of the tumor tissue decreased after radiotherapy and showed a significant difference between Pre-RT and RT 70 Gy, but not between Pre-RT and RT 50 Gy. The kep value decreased and Ve value increased after radiotherapy. The tumor regression rate was found to be positively correlated with the Ktrans value for Pre-RT (P=0.005) but negatively with the Ktrans value for RT 50 Gy (P=0.001). During the follow-up for 3 years, 5 patients died and 3 patients had distant metastases. No statistical differences in Ktrans, kep, or Ve were found between the groups with different prognosis. Conclusions The kinetic parameters in DCE-MRI, which vary significantly during radiotherapy, allow monitoring of tumor angiogenesis and vascular permeability and quantitative assessment of treatment efficacy for NPC. Ktrans value for Pre-RT and RT 50 Gy can serve as an indicator for early efficacy assessment of radiotherapy and for treatment adjustment, but its relation with the long-term outcomes awaits further study.

OBJECTIVETo investigate the feature of phenylalanine hydroxylase (PAH) gene mutations and provide guidance for genetic and prenatal diagnosis of patients with phenylketonuria from Shaanxi.

METHODSFor 55 patients whose blood Phe concentration was over 2.0 mg/dL, potential mutations in 13 exons and flanking sequences of the PAH gene were detected by PCR and DNA sequencing.

RESULTSA total of 98 mutations were detected in 110 PAH alleles, with the detection rate being 89.10%. Nine mutations have been identified in exon 7, which accounted for 33.67% of all. Exon 12 (14.29%) and exon 3 (12.24%) have followed. Thirty eight mutations, locating in exon2-exon12 and the flanking sequence, were detected in the 55 PKU patients. p.R243Q (24.49%) was the commonest mutation, whilstp.A47E, p.I65S and p.A259T were first discovered in China. After querying international databases including PAHdb and HGMD, the p.C334X was verified as the novel PAH gene mutation.

CONCLUSIONThe mutation spectrum of the PAH gene in Shaanxi has been identified. And a novel mutation has been identified. This may facilitate the diagnosis of PKU in the future.

Alleles ; Base Sequence ; Child ; Child, Preschool ; China ; Female ; Humans ; Infant ; Infant, Newborn ; Male ; Mutation ; Phenylalanine Hydroxylase ; blood ; genetics ; Phenylketonurias ; enzymology ; genetics

OBJECTIVETo determine the type and frequency of phenylalanine hydroxylase gene (PAH) mutations in ethnic Hui patients from Ningxia with phenylketonuria (PKU).

METHODSFor 35 PKU children patients and 50 healthy individuals, all exons and promoters of the PAH gene were analyzed with PCR and direct sequencing.

RESULTSTwenty mutations, including 8 missense mutations (40%), 5 nonsense mutations (25%), 4 splice site mutations (20%) and 3 deletion mutants (15%) were discovered. The overall detection rate was 68.57% (48/70). Common mutations have included R243Q (12.86%), R241C (11.43%), EX6-96A to G (5.71%), Y356X (5.71%), R413P(4.29%) and Q232X(4.29%), whilst rarer ones have included S16fsX10 (2.86%), R111X (2.86%) and L430P (2.86%). Among these, S16fsX10, L430P, D222G and IVS11+ 1G to A have not been reported previously. Y414X and S303fsX38 have not been reported in Hui ethnic group. No mutation was detected in the 50 normal controls.

CONCLUSIONThe types and distribution of PAH gene mutations in ethnic Hui from Ningxia have been different from other areas of China. The mutations also showed a rich diversity.

Adolescent ; Asian Continental Ancestry Group ; genetics ; Child ; Child, Preschool ; Female ; Humans ; Infant ; Infant, Newborn ; Male ; Mutation ; Phenylalanine Hydroxylase ; genetics ; Phenylketonurias ; enzymology ; genetics

Objective To understand the type and frequency of the gene mutation in exon 6 of phenylalanine hydroxylase(PAH) in the children of Ningxia,in order to provide foundation for phenylketonuria(PKU) gene diagnosis and prenatal diagnosis.Methods The exon 6 and flanking introns of 73 cases of classic PKU patients in Ningxia[all confirmed at Ningxia Neonatal Screening Center from Jan.2010 to Jun.2013,and distributed in Ningxia 22 County (city,district),aged from 15 days to 13 years,including 38 male cases,35 female cases,and Hui 39 cases,Han 34 cases] as well as 100 healthy newborn babies(Hui 50 cases;Han 50 cases) were sequentially analyzed by using the approach of PCR direct sequencing.Results There were 6 kinds of mutations detected,including EX6-96A ＞ G(6.85％),Q232X(2.74％),D222G(1.37％),V2301 (1.37％),R176X (0.68％) and N223I (0.68％).Mutation detection rate of exon 6 was 13.70％,and there were 3 mutation types:50.0％ missense mutation (3 types) ;33.3 ％ nonsense mutation (2 types) ;16.7％ cleavage site mutation(1 type).After reviewing the previous studies,the researchers had found out that EX6-96A ＞ G,Q232X and R176X were ever reported in China,and V2301 and D222G had been reported in our country for the first time but N223I was a new kind of PAH gene mutations were not been reported in the world.Conclusions It has defined the gene type and frequency of PAH gene mutations in exon 6 in the children of Ningxia and it will enrich the research of PKU in this area,and provide the basis for the development of gene diagnosis of PKU.