Objectivo To analyze urine organic acids in the urine using gas chromatography-mass spectrometry(GC/MS)for diagnosis of inherited metabolic diseases,especially for organic acids metabolic disorders.Methods 195 clinical urine samples from the patients with suspected organic acids metabolic disorders and 5 normal urine from adults were collected.After mixing some urine with intemal standards according to the concentration creatinine and adding hydroxylamine hydrochloride to mixture,the organic acids with hydroxyl group were oximated to the ketobodies.Organic acids were extracted twice with ethyl acetate and ethyl ether and derivatized with BSTFA-TMCS.An the organic acids were determined with Agilent GC/MS 6890/5973i with scan model.the mass-to-charge ratio range is 50-550 m/z,all data were nalyzed with Agilent GCMSD ChemStationG1701DA.We also investigated the linearity, accurate,precision.recovery and Carry-over by determining the internal standards in normal samples and positive organic acids in spiked control samples.Results More than one hundred kinds of organic acids in urine samples can be analyzed with this method.According to the two internal standards in normal urine samples,the minimal detection limit MMA and 2.PA was 2.5-2.8 μmol/L.Intra-and interassay coefficient of variation for MMA and 2-PA are both less than 10%.Pre-processing Interassay coefficient by sequential preparations of the same sample was 14%.The recoveries of the spiked samples were 95%-105%.Carryover analysis was less than 1%.All the parameters meet the requirement for clinical diagnosis.12 samples demonstrated positive including 6 cases of methylmalonic acidemia,1 case of propionic acidemia,3 cases of tyrosinemia Ⅰ,1 case of maple syrup urine disease and 1 cases of ketosis.Conclusions The method for the determination of organic acids in urine by GC/MS has been successfully established.It can be used for clinical screening and diagnosis for inherited genetic metabolic diseases.

Objective To explore the histocompatibility and plasticitity of hydroxyapatite/high-density polyethylene composite (HMPE). Design Experimental study. Participants Forty-five rabbits. Methods Rabbits were randomly devided into HA/PE, HA and PE surgical groups, every group included 15 rabbits. Osteoeetomy of orbital rim and implantation (HA/PE, HA and PE materials were used) were performed on 3 groups of the rabbits. At 1st week, 4th week, 8th week, 12th week and 24th week of postoperation, we took out the samples from the rabbits separately in each group and did histopathologic and electron microscope examinations. Main Outcome Measures Physical signs of the implant combination with the bone. The bone cell appeared in the implants. Results After 12 weeks, the calcium salts deposit like bone plate with some osteoblast inside the HA/PE implants was observed. After 8 weeks, the calci- um salts deposit inside the HA plants was observed. In the PE implants, nothing could be seen but some fibrous connective tissue. Conclusion HMPE is an ideal repair material especially for orbital bone.

AlM:To observe the efficacy of using botulinum toxin A in the treatment of blepharospasm.
METHODS: Totally 113 patients with blepharospasm were managed with a local injection of botulinum toxin A, and the therapeutic effect was evaluated.
RESULTS:Fifty-nine cases ( 52. 2%) had a complete remission of symptoms, 49 patients ( 43. 4%) presented with obvious relieved spasm, 4 cases ( 3. 5%) were partially relieved and the 1 patient ( 0. 9%) remained unchanged. The total effective rate was 99. 1%. The time of beginning effect was 1-14d. The recover time was mostly in 14d. The average of therapeutic effect lasted 1-9mo. Adverse reactions such as mild palpebra dysraphism, palpebra ptosis and local subcutaneous blood stasis were found in 23 patients, and the symptoms disappeared in 2-4wk.
CONCLUSlON:Botulinum toxin A can effectively control medium and severe blepharospasm by injecting a little dose on local muscle.

OBJECTIVE:To optimize ultrasonic extraction and purification technology of solanesol from tobacco leaf. METH-ODS:Using extraction rate and transport rate of solanesol as indexes,single factor test was used to investigate liquid-solid ratio, ultrasonic extraction temperature and time,ultrasonic power and extraction times,and the amount of soap alkali lye(volume ratio of soap alkali lye to extraction liquid),acidizing fluids (volume ratio of acidizing fluids to soap alkali lye extract),extraction times of purification technology. Optimized technology was validated,and the purity of solanesol was calculated;the amount of ex-tracted solanesol was compared between this method and traditional extraction method (spending 30 h),solvent continuous cyclic extraction (spending 5-6 h). RESULTS:Optimized extraction technology was as follows as volume ratio of soap alkali lye to ex-traction liquid 1∶14,ultrasonic extraction temperature 70 ℃,ultrasonic extraction time 60 min,ultrasonic power 120 W,extract-ing for 3 times;optimized purification technology was as follows as volume ratio of soap alkali lye to extraction liquid 2∶35,vol-ume ratio of acidizing fluids to soap alkali lye extract 2∶14,extracting for 4 times. In validation test,extraction rate,transport rate and purity were 92.45%(RSD=0.46%,n=3),79.88%(RSD=0.30%,n=3)and 55.86%(RSD=0.40%,n=3). The amount of solanesol extracted with 3 methods were 52.22,45.22 and 26.10 mg/g. CONCLUSIONS:The optimized technology is simple and stable,costs less time and saves source with high extraction amount and purity,which is suitable for production,extraction and purification of solanesol from tobacco leaf.

Objective To optimize the best subculture cycle through studying the influence of subculture time on tube plantlet growth of Dendrobium huoshanense and the change of medium composition.Methods Height,tiller,fresh weight,and chlorophyll content of the tube plantlet and pH value,water content,and sugar content of the medium were measured after different cycles of the subculture,the cost of culture medium for subculture was calculated as well.Results The height of the tube plantlet increase 282.86%,the tiller increase by 3.5 times,fresh weight reaches its maximum,chlorophyll content of the tube plantlet almost reaches its maximum after 40 d subculture;while water content and sugar content of the medium are decreased to the lowest point,pH value of medium(

Objective To analyze the change of serum levels of neuron‐specific enolase (NSE) and C reactive protein (CRP) and their early diagnostic value in hand‐foot‐mouth disease (HFMD) complicating encephalitis .Methods One hundred and twenty cases of HFMD and 50 healthy children(healthy control group) served as the research subjects and the HFMD cases were divided into the common HFMD group (n=70) and HFMD complicating encephalitis group (n=50) according to the clinical manifesta‐tions .The enterovirus 71 (EV71) in throat swab was detected by quantitative PCR .The NSE and CRP levels were detected by en‐zyme linked immunosorbent assay (ELISA) ,and white blood cell (WBC) count was measured by hematology analyzer .The NSE and CRP levels were compared and their diagnostic values were analyzed .Results The serum NSE and CRP levels in the HFMD complicating encephalitis group were higher than those in the HFMD common group and control group ,the differences were statis‐tically significant (P< 0 .05) ,and which in the EV71 positive group were significantly higher than those in the EV71 negative group ,the differences were statistically significant (P<0 .05) ,but WBC count had no statistically significant difference (P>0 .05) . The serum NSE level was positively correlated with the CRP level (r=0 .43 ,P<0 .01) .The area under ROC curves (AUC) and 95% CI of NSE and CRP were 0 .893(95% CI:0 .833 -0 .952) and 0 .867(95% CI:0 .799 -0 .934) ,the optimal operating points (OOP) were 11 .6 ng/mL and 14 .15 mg/L respectively ,the sensitivity and specificity of NSE and CRP for diagnosing HFMD com‐plicating encephalitis were 80 .0% ,86 .00% and 81 .4% ,78 .6% respectively ,while which of their combined detection were 88 .0%and 85 .7% ,AUC and 95% CI was 0 .927(95% CI:0 .845-0 .969) .Conclusion The NSE and CRP levels in children patients with early HFMD complicating encephalitis are significantly increased ,especially which in the patients with EV71 positive is more signif‐icant .The combined detection of serum NSE and CRP levels can be used as the early sensitive indicators for diagnosing HFMD complicating encephalitis .

Objective To prepare Exosomes secreted by mouse hepatoma cell (H_(22)) and heat stressed Exosomes (HS-Exo) derived from heat stress-treated mouse hepatoma cell (H_(22)), in order to study the possible anti-tumor immune mechanism. Methods Exosomes and HS-Exo were purified by serial ultracentrifugation and sucrose density gradiant centrifugation, and were observed and identified by electron microscope. The components and production of the protein and the effects of the host immune response against hepatocellular carcinoma of HS-Exo were observed by using Exosomes as the control. Their immunological factors were detected by Western blot. Lymphocyte proliferation and specific cytotoxic activity of mouse splenic cells were determined by MTT. CD4~+ and CD8~+ lymphocytes infiltration in mouse tumor tissues immunized by both were analysed by immunohistochemical staining. Results HS-Exo was similar in morphology to the Exosomes, the important immune-ralated protein expressed in HS-Exo was increased (P<0.05). HS-Exo immunized mouse group showed more effective inhibition of tumor growth, better-induced lymphocyte proliferation,more significantly enhanced the cytotoxic activity of spleen lymphocytes, as well as a more prominent role in tumor therapy than Exosomes immunized mouse control group (P<0.05). Conclusions Heat stress treatment method for the preparation of HS-Exo was feasible. HS-Exo had a stronger role in the immuneactivity and tumor treatment than control Exosomes.

Objective:To investigate the efficacy of sequential therapy of CD19 and CD22 chimeric antigen receptor T cells (CAR-T) for relapsed/refractory mediastinal B lymphoblastic lymphoma (B-LBL).Methods:One patient with relapsed/refractory mediastinal B-LBL treated with sequential therapy of CD19 and CD22 CAR-T who was admitted to Tongji Hospital of Huazhong University of Science and Technology Tongji Medical College in March 2017 was reported. At 1, 3, 6, 12 and 18 months after CAR-T therapy, the indicators of primary disease remission were monitored and the relevant literature was reviewed.Results:The patient relapsed and progressed after third-line chemotherapy, and then received sequential therapy of CD19 and CD22 CAR-T. In the course of cellular immunotherapy, the patient presented grade 1 cytokine release syndrome. After active treatment, the patient got stable condition and was discharged. The patient came to the hospital for regular review, and the mediastinal mass of the patient was dynamically followed up. After CAR-T therapy, the mediastinal mass of the patient was significantly reduced, and the patient was in continuous remission for 18 months.Conclusions:Sequential therapy of CD19 and CD22 CAR-T provides a new therapeutic approach for relapsed/refractory B-LBL. For patients with poor curative effect of conventional chemotherapy, CAR-T therapy should be actively performed as soon as possible to improve the remission rate and the long-term prognosis of patients.

Nowadays the role of genetic findings in determining the diagnosis, therapy and prognosis of acute myeloid leukemia (AML) has become more valuable. To improve and validate the detection of clonal chromosomal aberrations in leukemia, we designed a combined application of karyotyping with multiplex reverse transcription-polymerase chain reaction (RT-PCR) and fluorescence in situ hybridization (FISH), and addressed the expression and distribution of fusion genes among the subtypes of Chinese adult patients with de novo AML. Multiplex RT-PCR assays were performed on 477 samples from newly diagnosed AML patients, and cytogenetic data were obtained from 373 of them by R or G banding techniques and those in some cases were confirmed by FISH. The PCR products in some suspected cases were tested by two-directional sequencing. The results showed that except unqualified samples, fusion genes were detected by multiplex RT-PCR in 211 of 474 patients (44.51%), including AML1-ETO, CBFβ-MYH11, PML-RARα, PLZF-RARα, NPM-RARα, MLL rearrangements, BCR-ABL, DEK-CAN, SET-CAN, TEL-PDGFR, TLS-ERG, AML1-MDS1 (EVI-1). In 373 patients, who took both multiplex RT-PCR and karyotype analysis, the detection rate of chromosomal aberrations by using multiplex RT-PCR and karyotyping was 160/373 (42.89%) and 179/373 (47.98%) respectively, and the combination could optimize the detection rate of clonal genetic abnormalities to 216/373 (57.90%). The PCR results from 11 cases "normal" in karyotyping but abnormal in RT-PCR for MLL rearrangements were confirmed by two-directional sequencing. It is concluded that karyotype studies remain the cornerstone for genetic testing; conventional cytogenetics and molecular-based methods are complementary tests for the detection of clonal genetic aberrations in AML, especially for the cryptic or submicroscopic aberrations. Once a genetic marker has been identified by combined analysis, it could be used to monitor residual disease during/after chemotherapy, by quantitative RT-PCR and/or FISH.

Objective To investigate the effect of combined double-stranded RNA (dsRNA) and imiquimod stimulation on uterine immune cells. Methods In BALB/c × C57BL/6 mice and non-obese dia-betic (NOD) × C57BI/6 mice, embryo resorption rate was detected in the presence or absence of Toll-like receptor 3 (TLR3) agonist dsRNA [poly( 1: C)], TLR7 agonist imiquimod ( R837), or their combination, respectively. In in vivo system, the status of intracellular cytokine production in uterine CD45 + cells was de-tected by flow cytometry. To identify the CD45 + cells, uterine CD3+ T cells and CD49b + NK cells derived from placenta and decidua basalis were stimulated with dsRNA and imiquimod in in vitro systems, and the status of intracellular cytokine production was detected. Mitogen-activated protein kinase (MAPK) antago- nists SP600125 and PD98059 were used to block the increase of cytokine production. Results A synergistic increase of embryo resorption was observed after the induction of dsRNA and imiquimod combination. Mean-while, a synergistic increase of TNF-α and IFN-γ production was detected after the induction in CD45 + cells. Further study found that although synergistic effect can be detected in both CD3 + cells and CD49b + cells in BALB/c mice, the status was different in NOD mice. The cytokine increase should mainly be attrib-uted to CD3 + T cells, since no such increase was detected among the CD49b + NK cells in the NOD mice. The synergistic effect of combined agonists was partially inhibited by Jun N-terminal kinase (JNK) MAPK inhibitor SP600125 and almost completely abrogated by extracellular signal-regulated kinase (ERK) MAPK inhibitor PD98059. Conclusion Boosted TLR3 and TLR7 signal may be transmitted via Thl-type T cells, rather than NK cells in NOD mice. ERK MAPK pathway may be critical in TLR3 and TLR7 involved signa- ling.