Multicellular spheroid (MCS) can simulate many aspects of the in vivo physiological and pathological conditions in many aspects,better reflect the in-vivo behavior of cells in tumors.So it's increasingly accepted as a valuable tool for evaluating the efficacy of therapeutic intervention including chemotherapy,radiotherapy,immunotherapy and combined therapy.Various spheroid co-culture approaches have been presented to study heterologous cells interaction in solid tumors.The present review briefly introduces the methodology and applications of MCS with focus on the up-to-date information.

Tropane alkaloids,such as hyoscyamine (or its more stable racemate atropine) and scopolamine,are remembered as the oldest drugs in medicine with wide pharmaceutical applications clinically for their mydriatic,antispasmodic,anticholinergic,analgesic and sedative properties.Presently,the supply of tropane alkaloids are entirely rested on their isolation from plant materials,yet the low contents of tropane alkaloids in plants raise the requirement to breed new varieties of high-yield tropane alkaloids and improving the production of tropane alkaloids in hairy root cultures or plants by biotechnology has be a research focus in the field of secondary metabolism.For the past decade,there have been important progresses on biosynthesis of tropane alkaloids and their molecular biology,some new pathway genes have been identified and many achievements have also been acquired in metabolic engineering of tropane alkaloids.This review summarized the recent advances in above aspects,and the problems and orientations of future research are also discussed and proposed.

Objective: To establish an accelerated solvent extraction(ASE)-HPLC method to determine chrysophanol and aurantio-obtusin in Cassia obtusifolia L.Methods: The optimal extraction conditions were defined by orthogonal tests using ASE.The method was carried out on an ACE Excel C18-PFP column (75 mm×2.1 mm,2.5 μm) with the mobile phase consisting of 0.1% phosphoric acid solution-acetonitrile with gradient elution.The column temperature was 40 ℃,the flow rate was 0.4 ml·min-1, and the detection wavelength was 284 nm. Results: The best process parameters of ASE were as follows:the extraction solvent was methanol, the extraction temperature was 120 ℃ and the static extraction duration was 5 minutes for three cycles.The ASE method needed only 1/9 of the time as the pharmacopoeia method,while the extraction efficiency of the ASE method was higher.The linear ranges of cassia obtusifolia L.and Chrysophanol were at 0.73~58.57 μg·ml-1(r=0.999 7) and 1.09~87.29 μg·ml-1(r=0.999 6).The average recoveries were 102.7%(RSD=0.8%) and 98.2%(RSD=1.5%).Conclusion: The method is simple, rapid and sensitive, which can be used for the rapid determination of aurantio obtusin and chrysophanol in Cassia obtusifolia L.

Objective To improve the diagnostic accuracy of ovarian endometrioma and to discriminate it from other adnexal masses by analyzing its CT manifestations. Methods The CT images of 57 patients with 77 ovarian endometriomas proved by surgery and pathology were studied retrospectively.Results The cysts ranged in diameter from 2 to 14 cm with the mean size of 6.2 cm?5.1 cm. The margins of the cyst were sharp in 39 and ill defined in 38 endometriomas. Thin and thickened walls were found respectively in 40 and 37 of 77 endometriomas. Unilocular, bilocular and multilocular forms were seen respectively in 29(37.7%), 23(29.9%) and 25(32.5%) of 77 endometriomas. Gourd and sister forms were present respectively in 15 and 8 of 23 bilocular cysts, and sister loculi, outer son loculi, inner son loculi, and mixed forms in 10, 9, 1 and 5 of 25 multilocular endometriomas. The density of the cyst fluid varied from 11～65 HU with the mean of 28.5 HU. New hemorrhage inside cyst was found in 6 cysts. Slight or intermediate enhancement of the cyst wall of endometriomas in 42, and significant enhancement in 35 were demonstrated. 54 cysts, 13 cystadenomas with 18 lesions and 4 malignant tumors with 5 lesions were diagnosed by preoperatively by CT. The accurate rate of CT characterization was 71.4%. Conclusion The certain morphologic characteristics of the ovarian endometrioma can be showed by CT in higher extent. It is critical to familiar with these characteristics to improve the diagnostic accuracy, and to avoid misdiagnosis.

Objective To analytically screen the volatile components from Chenxiang Huaqi Tablets (CHT) using gas chromatography-triple quadrupole mass spectrometry (GC-MS/MS) combined with automated mass spectral deconvolution, and establish the methods of multiple components contents (camphor, bornyl acetate, and patchouli alcohol) from CHT using multi reaction monitor (MRM). Methods The chromatographic column was HP-5MS (30 m × 0.32 mm × 0.25 μm). Temperature program: the initial temperature was 100 ℃, at 15 ℃/min up to 300 ℃ for 2 min. Injection port temperature was 280 ℃; Ionization methods: EI ion source with ion source temperature of 230 ℃; Quadrupole temperature was 150 ℃; Interface temperature was 280 ℃; Inject volume was 1 μl. Results A total of thirteen components were screened by automated mass spectral deconvolution soft. Under the conditions of MRM collection, camphor, bornyl acetate, and patchouli alcohol were in the range of 4.5-90 mg/mL (r = 0.999 8), 3.3-66 mg/mL (r = 0.999 8), and 2.6-51.5 mg/mL (r = 0.999 9), respectively, the ratio of each component concentration to its peak area was linear, and the average recovery (n = 6) was 101.15%, 102.64%, and 100.10% respectively; The mass fraction of 10 batches of samples were in the ranges of 0.278-0.311, 0.381-0.438, and 0.229-0.381. Conclusion The method is accurate, simple, and good repeatability. It can be used for the simultaneous determination of camphor, bornyl acetate, and patchouli alcohol in CHT, which provides a reference for the improvement of the quality of this variety.

Objective To evaluate mivacurium-induced release of histamine in the patients undergoing general anesthesia.Methods Eighty patients of both sexes,aged 19-58 yr,weighing 42-75 kg,of ASA physical status Ⅰ or Ⅱ,undergoing ureteroscopy under general anesthesia,were randomly divided into 2 groups (n =40 each) using a random number table:rocuronium group (group R) and mivacurium group (group M).Anesthesia was induced with iv midazolam 0.04 mg/kg,fentanyl 3 μg/kg,etomidate 0.3 mg/kg,and mivacurium 0.20 mg/kg (group M) or rocuronium 0.75 mg/kg (group R).Streamlined liner of the pharynx airway (SLIPA) was inserted for mechanical ventilation.PET CO2 was maintained at 35-40 mmHg.Anesthesia was maintained with fentanyl and propofol.Before muscle relaxant administration,at 3 min after muscle relaxant administration and at 5 min after insertion of SLIPA,venous blood samples were collected for determination of plasma histamine concentrations (by HPLC) and the histamine release was classified.Results Compared with group R,the plasma histamine concentration and histamine release were increased at 3 min after muscle relaxant administration in group M.No fatal responses caused by histamine release were found in the two groups.Conclusion Although mivacurium induces release of histamine,it can still be safely used for the patients undergoing ureteroscopy under general anesthesia.

Esophageal Neoplasms ; metabolism ; pathology ; Female ; Humans ; Melanoma ; metabolism ; pathology ; Melanoma-Specific Antigens ; metabolism ; Middle Aged ; S100 Proteins ; metabolism

Hyoscyamine 6 beta-hydroxylase (H6H) is the last rate-limiting enzyme directly catalyzing the formation of scopolamine in tropane alkaloids (TAs) biosynthesis pathway. It is the primary target gene in the genetic modification of TAs metabolic pathway. Full-length cDNA and gDNA sequences of a novel H6H gene were cloned from Datura arborea (DaH6H, GenBank accession numbers for cDNA and gDNA are KR006981 and KR006983, respectively). Nucleotide sequence analysis reveals an open reading frame of 1375 bp encoding 347 amino acids in the cDNA of DaH6H, while the gDNA of DaH6H contains four exons and three introns, with the highest similarity to the gDNA of H6H from D. stramonium. DaH6H also exhibited the most identity of 90.5% with DsH6H in amino acids and harbored conserved 2-oxoglutarate binding motif and two iron binding motifs. The expression level of DaH6H was highest in the mature leaf, followed by the secondary root, and with no expression in the primary root based on qPCR analysis. Its expression was inhibited by MeJA. DaH6H was expressed in E. coli and a 39 kD recombinant protein was detected in SDS-PAGE. Comparison of the contents of scopolamine and hyoscyamine in various TAs-producing plants revealed that D. arborea was one of the rare scopolamine predominant plants. Cloning of DaH6H gene will allow more research in the molecular regulatory mechanism of TAs biosynthesis in distinct plants and provide a new candidate gene for scopolamine metabolic engineering.

BACKGROUND:Sirius red is a strong acid anionic dye. Being not-easyto-fade and specific, sirius red becomes the best dye for collagen staining.Collagen is a major component of extracellular matrix and has some specific physiological functions. Through synthesis and reconstruction of collagen, bone fracture repair will be accomplished.OBJECTIVE: Picric acid-Sirius red stained slides were observed under a polarized light microscopy for evaluation the dynamic changes in the ratio of different collagen types and their distributions in bone fracture healing.DESIGN: It was a controlled observation.SETTING: It was conducted in the Department of Orthopedics, Renmin Hospital, Wuhan University; Department of Traumatic Orthopaedics, Tianjin Hospital; Department of Traumatic Orthopaedics, Jishuitan Hospital,Medical Department, Peking University; Tissue Engineering Center of Institute of Basic Medical Sciences, Academy of Military Medical Sciences of Chinese PLAMATERIALS: It was conducted at Tissue Engineering Center of Institute of Basic Medical Sciences, Academy of Military Medical Sciences of Chinese PLA from March 2002 to September 2003. Three healthy adult Chinese sheep, male and in weight from 25 to 35 g, were selected.METHODS: All the animals were anesthesized and sterilized; a transverse osteotomy of the trunk of metatarsus was performed; and the end of fracture was fixed with a six-hole Medoff sliding plate. At the post-operative month 1, 3 and 6, samples were taken from bone fractures. After decalcification with EDTA, they were stained with Picric acid-sirius red, and the types and distribution of collagens were observed under a polarized light microscopy.MAIN OUTCOME MEASURES: Types and distributions of collagens in bone lesion in different period of bone healing were investigated.RESULTS: Three sheep used in this study entered the statistical analysis.①Morphological features of various collagens under a polarized light microscopy postoperatively: Type Ⅰ collagen packed tightly, with a strong refraction and yellow, orange or red thick fibres. Type Ⅱ collagen formed a loose reticulation with fibres exhibiting different colour and a weak refraction. Thin fibres of type Ⅲ collagen with weak refraction and green colour formed a loose reticulation. ②Quantitative studies on various collagens under a polarized light microscopy postoperatively: At postoperative month 1,red or orange fibres (type Ⅰ collagen) were rarely seen in bone fracture,while green fibres (typical of type Ⅲ collagen) were dominant with a disorder pack. At postoperative month 3, red or orange fibres increased significantly and the ratio of type Ⅲ collagen reduced. The collagen fibres assembled regularly. At postoperative month 6, thick yellow-red collagen became dominant and thin green type Ⅲ collagen decreased dramatically and arranged in an obvious oblique, spiral and crossed orientations.CONCLUSION: Picric acid-sirius red stain combined with polarized light microscopy technique is not only capable of identifing type Ⅰ and type Ⅲ collagens in bone fraction, but also can reflect the morphological features,distribution and the ratio of these two type collagens. This approach has the virtues of easiness in operation, strong specificity and high sensitivity.

Objective To investigate the value of multi-slice CT (MSCT) in diagnosing early acute appendicitis (AA).Methods From June 2008 to June 2011,abdomen MSCT images of 41 patients with acute simple appendicitis confirmed by surgery and pathology were evaluated retrospectively. Thirty-six patients with clinically confirmed normal appendix served as the control groups with 18 patients in complicated-normal-appendix (CNA) group and 18 patients in noncomplicated-normal-appendix (NCNA)group. The appendix was reconstructed by using multiplanar reformation (MPR) and curved planar reformation (CPR) techniques. The differences between early AA and normal appendix in appendiceal diameter,thickness of the appendiceal wall, maximum depth of the intraluminal appendiceal fluid (MDIAF), abnormal enhancement of the appendiceal wall, appendicolith and the periappendiceal abnormalities were evaluated and compared by using analysis of variance,R test and Chi-square test.Results The mean thickness of the appendiceal wall was (2.88 ±0.62),(2.58 -±0.50) and (2.73 ±0.53) mm in early AA,CNA and NCNA groups respectively,with no statistically significant difference among them ( F =1.73,P=0.19).The nean appendiceal diameter was (11.37 ± 1.94),(7.03 -±0.89),(6.75 ±0.63) mm,and median MDIAF was 4.05 (2.65-8.50),1.68 (0-.40),0 (0-1.90) mm in early AA,CNA and NCNA groups respectively,with statistically significant differences between early AA and the two normal appendix groups ( Z =7.02,7.24 ; P =0.00 ).The abnormal enhancement of appendiceal wall was found in 61.1％ (11/18) of early AA,16.7％ (3/18) of CNA and 11.1％ (2/18) of NCNA groups respectively,with statistically significant differences between early AA and the two normal appendix groups (x2 =12.83,P =0.00). Using a cutoff value of 7.8 mm of the appendiceal diameter and 2.6 mm of MDIAF for the early AA,the sensitivity,specificity and accuracy were 97.6％ (40/41),91.7％ (33/36) and 94.8％ (73/77),and 100.0％ (36/36),88.9％ (32/36) and 94.4％ (68/72),respectively.Conclusions MSCT is particularly useful for the diagnosis of early AA. When appendiceal diameter is greater than 7.8 mm,and MDIAF greater than 2.6mm,early AA can be diagnosed with confidence.