Analysis of Gene Mutation Types of Thalassemia in Fuzhou Area of China / 中国实验血液学杂志

OBJECTIVE@#To investigate the gene mutation types and spectrum of α, β-thalassemia in Fuzhou area of China.@*METHODS@#Thalassemia gene screening was performed in the women receiving physical, prenatal, and pre-pregnancy examination, and the patients with suspected thalassemia in our hospital from July 2013 to March 2018.Genotypes of thalassem were detected by Gap-PCR and RDB-PCR.@*RESULTS@#1042 were positive among 2074 suspected cases with a positive rate of 50.24%; 618 cases were confirmed to be α-thalassemia and with a positive rate of 29.8%; 409 cases were confirmed to be β-thalassemia with a positive rate of 19.72%. 15 cases were confirmed to be α-β complex thalassemia with a positive rate of 0.72%. the --/αα(76.54%) was the most common genotype among α-thalassemia, -α/αα(10.03%) and -α/αα(2.91%) in hot pursuit. In addition, IVS-II-55 (T->G) and IVS-II-119 (-G, +CTCGGCCC) were newly found alpha mutations; the IVS-2-654 (C→T) (40.83%) was the most common genotype among β-thalassemia, CD41-42 (-TCTT) (35.94%) and CD17 (A→T) (9.78%) in hot pursuit.@*CONCLUSION@#The genotype of thalassemia in Fuzhou area is highly heterogenic, --/αα is the most common genotype among α-thalassemia, IVS-2-654 (C→T) is the most common genotype among β-thalassemia, Meanwhile, two α-mutation sites are found in this study which were not reported in the Database of Human Hemoglobin Variants and Thalassemias.

Non-surgical treatment of lumbar intervertebral disc protrusion without blood stasis:a serum proteomic analysis / 中国组织工程研究

BACKGROUND: Proteomics is a well studied research method, but its application in the non-surgical treatment of lumbar intervertebral disc protrusion (LIDP) is little reported. OBJECTIVE: To screen the differentially expressed proteins in patients with LIDP but without blood stasis before and after non-surgical treatment by proteomics. METHODS: Sixty patients with LIDP but without blood stasis were selected, and treated with non-surgical treatment for 4 weeks. The differentially expressed proteins were screened and identified by iTRAQ combined with LC-MS/MS. The bioinformatics analysis of the identified proteins was carried out, and the curative effectiveness was investigated. RESULTS AND CONCLUSION: Compared with those before treatment, the Visual Analogue Scale scores were significantly (P ＜ 0.05), the Japanese Orthopedic Association scores were significantly increased decreased (P ＜ 0.05), and the excellent and good rate reached 95.0% post-treatment. A total of 300 differentially expressed proteins were screened and 25 significantly expressed proteins were identified (P ＜0.05). Bioinformatics analysis revealed that nine of the significantly expressed proteins were enriched to 15 KEGG signaling pathways. These results suggest that the use of Western medicine non-surgical treatment for the LIDP without blood stasis can achieve satisfactory results. Besides, complement C1qA, cDNA protein (FLJ60724), complement C4B frameshift mutation, cDNA protein (FLJ53025), mannose binding protein C, apolipoprotein B, hemoglobin α-1 globin chain variant, hemoglobin β subunit and cDNA protein (FLJ76254) may be the potential serum markers of the non-surgical treatment for the LIDP without non-blood stasis.

Discovery and tracking source of the new subgroup of Chinese Francisella tularensis type B / 中华预防医学杂志

<p><b>OBJECTIVE</b>To perform laboratory diagnosis and tracking source of a suspected tularemia patient in Beijing.</p><p><b>METHODS</b>A suspected tularemia patient was reported in Beijing city on July 19, 2012. Genomic DNA was extracted from the blood sample of the patient, then general PCR and sequencing of amplicons were conducted using 3 specific genes (fopA, tul4 and 16S rRNA) Francisella tularensis (F.tularensis), and 2 genotyping primers (C1C4 and RD1). Two other laboratories repeated the PCR and sequencing of the fopA in parallel. At the same time, real-time PCR fluorescent ration was performed using 4 targets (fopA, ISFtul2, 23kDa, and tul4), and phylogenetic analysis was carried out using 11 canonical single nucleotide polymorphisms (SNPs) and 4 insertions or deletions.</p><p><b>RESULTS</b>All the 3 specific genes were amplified positively, and sequenced fragments were 409, 407 and 1 053 bp, respectively. The patient was infected by F. tularensis comparing with the whole genome published. Next, amplicons of 151 and 924 bp were obtained by the 2 typing primers after sequencing, respectively. The segment lengths suggested that the patient was infected by the subsp. holarctica. All of the two other laboratories obtained positive data for the PCR and sequencing of the fopA. In addition, all the 4 targets tested positive by real-time PCR for F. tularensis. The Ct value of the fopA, ISFtul2, 23kDa and tul4 were 30, 25, 28, and 30, respectively. The phylogenetic analysis indicated that the whole genome of this case was assigned to a known clade from Russia, which was subgroup B3.</p><p><b>CONCLUSION</b>This case was confirmed to be a tularemia patient, and a new subgroup of F. tularensis type B was found in China.</p>

Analysis of Factors Influencing the Generation of Unqualified Clinical Samples and Measures to Prevent this Generation

BACKGROUND: We investigated the influence of pre-analytical factors on the results of clinical tests and thereby analyzed approaches to improve quality management in clinical laboratories. METHODS: Unqualified clinical samples were selected from all the samples received at our clinical laboratory. The data were collected for 2009 and 2010, i.e., the years before and after the establishment of the laboratory quality management system. The rate and causes of generation of unqualified samples were analyzed, and measures to improve the laboratory practices were studied and implemented. RESULTS: A total of 1,051 unqualified samples were identified from among the 553,158 samples (the overall incidence rate of unqualified samples was 0.19%). The number of unqualified samples substantially varied according to the nature of the sample, and clinical samples collected for routine blood tests or coagulation tests were the predominant unqualified samples. The main causes of generation of unqualified samples were insufficient sample volumes and improper methods of mixing the samples. The rate of generation of unqualified samples decreased significantly after the implementation of improvement measures (0.26% in 2009 vs. 0.13% in 2010, P<0.001). CONCLUSIONS: The number of unqualified samples decreased significantly after the establishment of the laboratory quality management system, which promoted active communication among and training of the clinical staff to reduce the occurrence of pre-analytical errors. Comprehensive control of pre-analytical factors is an important approach in improving the clinical laboratory practices.

Silencing of the annexin II gene down- regulates the levels of S100A10, c-Myc, and plasmin and inhibits breast cancer cell proliferation and invasion

To explore the roles of annexin II in breast cancer progression, and to study the effect of annexin II on breast cancer cell proliferation and invasion. This study was conducted in the Key Laboratory of Diagnostic Medicine Designated by the Ministry of Education, Chongqing Medical University, Chongqing, China from December 2006 to January 2009. First, we employed Western blot and reverse transcriptase polymerase chain reaction to detect the expression of annexin II and S100A10 in a panel of well-characterized human breast cancer cell lines, and investigated the localization of annexin II and S100A10 by use of immunofluorescence. We then silenced the expression of annexin II in MDA-MB-435s, which was found to over express annexin II, using the chemically-synthetic annexin II small interfering RNA [siRNA] duplexes [including 3 groups: blank MDA-MB-435s cells, cells transfected with negative control siRNA, and cells transfected with annexin II-siRNA]. Finally, the cell proliferation, invasion, and plasmin generation were assayed, and the cellular levels of S100A10 and c-Myc were also detected. All the tests were repeated 3 times. Annexin II and S100A10 were over expressed in invasive human breast cancer cell lines. The siRNA targeting annexin II of MDS-MB 435s cells did not only decrease annexin II messenger RNA and protein levels, but also down-regulated the levels of S100A10, and c-Myc. The treated cells were remarkably blocked in the G0/G1 phase, and cells in the S/G2+M phase decreased. Additionally, the treatment with siRNA resulted in reduction of plasmin generation as well as a loss of the invasive capacity of breast cancer cells. Annexin II might be a key contributor to breast cancer proliferation and invasion

In vivo characterization of streptococcus pneumoniae genes involved in the pathogenesis of meningitis by differential fluorescence induction

To further understand the pathogenesis of pneumococcal meningitis, and provide some target candidates for the development of drugs. This study was performed at the Department of Laboratory Medicine, Key Laboratory of Diagnostic Medicine [Ministry of Education], Chongqing Medical University, Chongqing, China from March 2006 to December 2007. A promoter-trap library of Streptococcus pneumoniae TIGR4, reported by green fluorescent protein was constructed, and used to infect BALB/c mice [n=15] intranasally, to set up a meningitis model. The control group [n=5] were inoculated with sterile phosphate buffered saline. The bacteria containing the promoter fusions induced only in meningitis brain tissue, not in vitro were screened by differential fluorescence induction. The obtained bacteria were prepared to re-infect the mice and re-screened, as above. The sorted bacteria were spread on trypticase soy agar with 5% sheep blood agar plates containing chloramphenicol [2.5 micro g/ml], and were used for DNA cloning, sequencing, and bioinformatics analysis. A total of 52 genes were obtained. Bioinformatics analysis revealed that these in vivo induced genes were involved in functions such as, adherence, energy metabolism, nutrient substance transport, transcription regulation, DNA metabolism, as well as, cell wall synthesis. In addition, there were some genes encoding for some hypothetical proteins with unknown, or putative functions. Pneumococcal genes involved in meningitis identified in this study are potential targets to understand the pathogenesis of pneumococcal meningitis

Developmental variation of renal apparent diffusion coefficient in rat / 中华放射学杂志

Objective To analyze the changes of renal apparent diffusion coefficient(ADC)value during development in intact rats.Methods Five intact male Wistar rats(1 month of age)were involved in this study.Using SE-echo planar imaging(EPI)sequence to acquire renal DWI at a 3.0 T MR on day 1,day 5,day 10,day 30,and day 50,respectively.The b value was 0 and 500 s/mm2.The ADCs of the cortex and the medulla were measured on the right kidney and the renal volume was calculated by manually renal outling on each slice.The difference of ADC between the cortex and the medulla was analyzed using a paired student t test,and the changes of renal volume and ADCs with rats development were evaluated with a repeated measurement ANOVA.Results The ADC of the cortex was higher than that of the medulla except on day 1(P＜0.01).when b value 0 and 500 s/mm2 were chosen.Renal volume increased with the rat development.from(0.86±0.02)ml to(1.47±0.21)ml.And the ADCs of both the cortex and medulla increased from(1.66±0.14)×10-3mm2/s to(1.96±0.08)×10-3mm2/s for the cortex and from (1.54±0.12)×10-3mm2/s to(1.91±0.09)×10-3mm2/s for the medulla.Conclusion Renal ADCs of both the cortex and medulla increase during the period from 1 to about 3 months of age in rats.The influence of age on renal ADC should be considered when choosing rats aged from 1 to 3 months for MR study.

Reproducibility of measuring renal oxygenation by blood oxygen level-dependent MRI in rat / 中华放射学杂志

Objective To evaluate the reproducibility of measuring renal oxygenation in rat using blood oxygen level-dependent MR imaging(BOLD MRI).Methods Five intact male Wistar rats were included in this study and their body weights were measured before MRI scans.BOLD MRI were performed ou day 1(d1),day 5(d5),day 10(d10),day 30(d30)and day 50(d50)on a 3.0 T MR scanner to measure the apparent spin-spin relaxation(R2*).On d30 and d50,the differences of R2*(△R2*)were calculated between before and 10 min after furosemide injection through the tail vein.The reproducibility of the baseline R2* of 5 times repeated scans were verified by the repeated-measure ANOVA test.The R2* and △R2* between pre-and post-furosemide iniection on d30 and d50 were measured on the codex and the medulla and a paired t test was run to analyze their responses to furosemide and the reproducibility of △R2*.Results The average body weight on d1,d5,d10,d3 and d50 was(150.4±3.7)g、(170.2±7.0)g、(201.0±5.8)g、(306.2±17.0)g and(352.0±12.2)g,respectively,with statistical difference(F=422.103.P＜0.01).The R2* showed no statistical difference in the cortex and medulla among the five scans(P＞0.05).On d30.the R2* of the cortex and medulla was(25.2±1.2)and(32.8±2.2)Hz before and significantly decreased to(21.1±2.2)and(25.9±3.0)Hz after furosemide administration,respectively(P＜0.01).On d50,the R2* of the cortex and medulla was(25.9±0.8)and(34.3±3.9)Hz before and significantly decreased to(20.2±1.5)and(27.0±3.2)Hz after furosemide administration.respectively(P＜0.01).The △R2* on the codex and medulla was(4.1±1.7)and(6.9 4-2.8)Hz on d30 and(5.8±1.1)and(7.3±2.8)Hz on d50,respectively,but there is no significant difference between eortex and medulla(P＞0.05).Conclusion The baseline R2* in the cortex and medulla were reproducible over 50 days and they were not body weighted.On 3.0 T MR scanners.R2* in the codex and medulla decreased significantly after administration of furosemide.

Protection elicited by immunization of ClpP against Streptococcus pneumoniae infection in mice / 中华微生物学和免疫学杂志

Objective To obtain purified ClpP produced by prokaryotic expression system,and to evaluate the protection effect elicited by ClpP in animal protection test.Methods The template DNA was isolated from the culture of TIGR4 Streptococcus pneumoniae.The complete ClpP open reading frame(ORF)was cloned into pET-32a expression vector by gene recombination technology in vitro.After prokaryotic expression,purification and sequence identification,the recombinant ClpP were innoculated into mice,and at the same time a group of mice were inoculated with the antibody to ClpP.We monitored the survival time of the innoculated mice after being challenged intraperitoneally with,TIGR4.Results We obtained high-expressed recombinant antigen protein which was then identified by Western blot,and we have got the recombinant antigen protein with a purity of more than 90%after being purified through Ni2+ affinity chromatography and processed by dialysis.The sunrvival time of mice immunized with ClpP or ClpP antibody were significantly langer than that of the mice received PBS.Conclusion The recombinant ClpP antigen protein can elicit protection to the invasive S.pneumoniae infection in mice,which might make ClpP as a candidate of S.pneumoniae vaccine.

Correlation between Single Nucleotide of Polymorphisms of -238,-308 G/A in Tumour Necrosis Factor-? Gene Promoter and Juvenile Idiopathic Arthritis / 实用儿科临床杂志

Objective To study the correlation between single nucleotide polymorphisms of -238,-308 G/A in promoter region of tumor necrosis factor -?(TNF-?) gene and the type of juvenile idiopathic arthritis (JIA).Methods Clinical data and blood preparation of 127 children with JIA and 106 healthy children were evaluated.Subgroups of JIA were defined according to the Edmonton criteria.The -238 G/A and -308 G/A polymorphisms in DNA analysis in this study were extracted from the whole blood.The restricted fragment length polymorphisms were determined in the cases of all JIA children and control group.Results 1.The TNF-?-238 G/A allele frequencies of JIA group and control group:allele frequency of JIA group was 92.9% and 7.1%,and the control group was 95.3% and 4.7%.The distribution of allele frequencies was no significantly different between JIA group and control group(?2=1.149 P=0.284).But there were significant difference between polyarticular JIA (RF negative) and control group(?2=7.621 P=0.006).2.The TNF-?-308 G/A allele frequencies of JIA group and control group:allele frequency of JIA group was 94.1% and 5.9%,the control group was 95.3% and 4.7%.The distributions of allele frequencies was no significantly different between JIA group and control group(?2=0.322 P=0.571).There were significantly difference between polyarticular JIA (RF negative) and control group (?2=7.621 P=0.006).Conclusions The TNF-?-238,-308 polymorphisms of A in the-238 and-308 TNF-? gene are important to the joint destruction of JIA.The study will be beneficial to provide indirect support to the application of anti-TNF drugs to the treatment of JIA.

Prokaryotic expression, purification, identification of human cystatin C and preparation of its antiserum / 第三军医大学学报

Objective To construct a prokaryotic expression vector of cystatin C (Cys C), purify Cys C protein produced by the expression system, and prepare its antiserum. Methods Total RNA was isolated from HL-60 cells, and human Cys C gene was amplified with RT-PCR. The cDNA fragment was cloned into pMD18-T vector and which was confirmed by sequencing. The enzyme-digested target fragment was cloned into PET-32(a) expression vector and transfected into E.coli. BL 21(DE3), in which Cys C expression was induced. After the inclusion body protein was purified through Ni2+ affinity chromatography, processed by dialysis, identified by Western blotting, a rabbit was immunized with the fusion protein, and the antiserum was obtained. Results The result of DNA sequence analysis showed that the cloned Cys C gene sequence was completely corresponding to GenBank data. SDS-PAGE and Western blotting showed that the expressed Cys C fusion protein was about 35?103, mainly existing in the inclusion body of E.coli., that could be purified through Ni2+ affinity chromatography. The titer of the antiserum to the purified protein was 1∶8 000 by ELISA, and Western blotting confirmed that the antiserum reacted specifically to the Cys C protein. Conclusion A recombinant Cys C protein and the specific polyclonal antibody have been obtained, which provides a basis for establishment of immunoassays of human Cys C.