Tropane alkaloids (TAs) are a class of anticholinergic drugs widely used in clinical practice and mainly extracted from plant, among which Atopa belladonna is the main commercial drug source. It is of great industrial value to obtain TAs in large quantities by plant metabolic engineering. In TAs pathway, cytochrome oxidase CYP82M3 catalyze the synthesis of tropinone and then tropinone reductase I (TRI) compete with TRII for tropinone to form tropine leading to the TAs synthesis (drainage). In this study, based on the "increasing flow and drainage" metabolic engineering strategy, two genes, namely HnCYP82M3 and DsTRI from Hyoscyamus niger and Datura stramonium, respectively, were overexpressed in the hair roots of A. belladonna, with a view to promote the TAs accumulation. The HnCYP82M3 gene was cloned from the root of H. niger, and it encoded amino acid with 91.7% sequence identity with AbCYP82M3 from A. belladonna. Overexpression of HnCYP82M3 alone did not affect the content of TAs in hair roots of A. belladonna, indicating that CYP82M3 was not a key enzyme in TAs biosynthesis. Simultaneous overexpression of HnCYP82M3 and DsTRI greatly promoted the accumulation of the three TAs, and the contents of hyoscyamine, anisodamine and scopolamine were 4.97 times, 2.83 times and 2.19 times that of the control, respectively, and the increase amplitude was greater than that of single overexpression of DsTRI. This study showed that the "increasing flow and drainage" strategy of enzyme genes co-expression at branch points was a promising metabolic engineering method to effectively improve the biosynthesis of TAs in A. belladonna, and laid a theoretical and technical foundation for the large-scale industrial acquisition of TAs.

The rol genes on pRiA4 plasmid of Agrobacterium rhizogenes are potent genes that promote secondary metabolism. Molecular breeding of Atropa belladonna can be conducted by introducing rol genes to increase tropane alkaloids (TAs) content in A. belladonna. In this study, the rolB gene was overexpressed in A. belladonna plants to study the effect of rolB gene on the biosynthesis of TAs. The phenotype, TAs content and expression levels of key enzyme genes in the pathway of TAs biosynthesis of transgenic A. belladonna were analyzed. The results showed that transgenic A. belladonna had developed root system, enlarged leaves, increased leaf fresh weight, deepened leaf color, enlarged flowers, changed flower shape, reduced pistil height and decreased pollen vitality. The content of TAs in the stems of transgenic A. belladonna was significantly higher than that of the control, and the contents of scopolamine, anisodamine, hyoscyamine can reach 2.11-2.91, 1.23-2.37 and 4.88-5.20 times of the control, respectively. Compared with the control group, the expressions of key enzymes putrescine N-methyltransferase (PMT), type III polyketide synthase (PYKS), tropinone reductase I (TRI), aromatic amino acid aminotransferase 4 (ArAT4), UDP-glycosyltransferase 1 (UGT1) and hyoscyamine 6-β-hydroxylase (H6H) in the TAs biosynthesis pathway were up-regulated, and the expression of tropinone reductase II (TRII) as a metabolic shunting gene was down-regulated. The results indicated that rolB gene enhanced TAs synthesis ability in roots and accumulation in stems of A. belladonna by enhancing metabolic flow of TAs synthesis pathway and weakening the metabolic shunt of competing pathway. This study laid a foundation for molecular breeding of A. belladonna with high-yield TAs content using rolB gene.

ObjectiveTo further study the pathogenic role of different types of Chlamydia trachomatis （CT） proteins in tubal factor infertility， evaluate the clinical detection value of Chlamydia trachomatis protein antibody in predicting tubal factor infertility. MethodsA total of 58 cases of tubal factor infertility （TFI）， 41 cases of fertile controls （FC） and 18 cases of infertile controls （IFC） were included. For serum detection， first， CT-IgG ELISA kit was used to detect the expression of CT-IgG in serum of three groups of people； then， 6 kinds of Chlamydia trachomatis proteins were expressed and purified in the early stage to establish the antibody test for these proteins， and ELISA detection method was used to detect the expression of their antibodies in the serum of TFI group， FC group and IFC group， respectively； and finally， the antibody OD value of the 6 kinds of Chlamydia trachomatis proteins in the three groups of subjects were statistically described， and CT-IgG was used as the reference standard to draw the receiver operating characteristic curve （ROC curve） of each CT antibody. The Youden Index determines the cutoff value for each antibody. Taking TFI as the reference class， two disordered multiple classification logistic regression models were established with the FC and IFC groups， respectively； and the reference class was used to explore the value of various antibodies and age in predicting TFI， FC and IFC of Chlamydia trachomatis. The back-off method was used to screen the variables. ResultsThe OD value of CT376 antibody in the TFI group was higher than that in the FC group （0.86 vs. 0.60， P=0.026）. The CT376 antibody OD value in the TFI group was higher than that in the IFC group （0.86 vs. 0.64， P=0.026）. The CT443 antibody OD value in the IFC group was higher than that in the TFI group （0.59 vs. 0.34， P=0.036） and higher than that in the FC group （0.59 vs. 0.30， P=0.02）. The multiple classification logistic regression analysis established between TFI and FC showed that CT-IgG ［P<0.001， OR=0.084， 95%CI （0.025， 0.284）］， CT376 antibody ［P=0.068， OR=0.359， 95%CI （0.120， 1.078）］. CT-IgG is an independent risk factor for tubal infertility， and CT376 antibody cannot be an independent risk factor for tubal infertility. The multiple classification logistic regression analysis established between TFI and IFC showed that among infertile patients， CT-IgG ［P<0.05， OR=0.194， 95%CI （0.046， 0.817）］， CT376 antibody ［P<0.05， OR=0.176， 95%CI （0.038， 0.818）］ and CT381 antibody ［P<0.05， OR=0.112， 95%CI （ 0.016， 0.796）］ were independent risk factors for tubal infertility. ConclusionThe expression of CT376 antibody in tubal infertility patients is higher than that in fertile and infertile controls， suggesting that CT-induced tubal factor infertility may be related to CT376. CT-IgG， and CT376 antibodies are meaningful in predicting CT-induced tubal factor infertility.

INTRODUCTION: Chest physiotherapy (CPT) may benefit children aged below five years who suffer from lower respiratory tract infection (LRTI). However, its effects depend on the technique used. This study aimed to determine whether mechanical CPT using the LEGA-Kid® mechanical percussion device is superior to manual CPT in children with LRTI. METHODS: Children aged five months to five years who were admitted and referred for CPT from January to April 2017 were randomised to either manual CPT or mechanical CPT with LEGA-Kid. Outcomes measured before intervention and two hours after intervention were respiratory rate (RR), oxygen saturation and modified Respiratory Distress Assessment Instrument (mRDAI) score. RESULTS: All 30 enrolled patients showed significant reduction in post-intervention RR and mRDAI scores. There was an 8% reduction in RR for the manual CPT group (p = 0.002) and a 16.5% reduction in the mechanical CPT group (p = 0.0001), with a significantly greater reduction in the latter (p = 0.024). mRDAI scores decreased by 2.96 in the manual group (p = 0.0001) and 3.62 in the mechanical group (p = 0.002), with no significant difference between the groups. There was no significant improvement in oxygen saturation, and no adverse events were observed after CPT. CONCLUSION Children receiving both manual and mechanical CPT showed improvements in respiratory distress symptoms, with no adverse effects. A combined strategy of nebulised hypertonic saline followed by CPT for LRTI removes airway secretions and results in improvements in moderately severe respiratory distress. The LEGA-Kid mechanical CPT method is superior to manual CPT in reducing the RR.
Child ; Humans ; Percussion/methods* ; Physical Therapy Modalities ; Respiratory Distress Syndrome ; Respiratory Therapy/methods* ; Respiratory Tract Infections ; Single-Blind Method

OBJECTIVE: To investigate the effect of a noval radio-frequency atmospheric-pressure glow discharge (RF-APGD) plasma jet on crosslinking of dentin collagen. METHODS: (1) Twenty intact third molars were collected. The middle dentin discs were prepared for each tooth by low-speed water-cooled Isomet saw, and then immersed in 10% (mass fraction) H₃PO₄ solution for 16 h to obtain fully demine-ralized dentin collagen. The twenty dentin discs were then randomly divided into five groups. The control group was untreated while the four experimental groups were treated by plasma jet with gas temperature of 4 ℃ for different times (20 s, 30 s, 40 s, and 50 s). The structure and crosslinking degree of dentin collagen were characterized by attenuated total reflection-Fourier transform infrared spectroscopy. The surface morphology of demineralized dentin was observed by scanning electron microscope, and the microstructure was observed by transmission electron microscope. (2) Fourty non-caries third molars were collected to prepare 5 g fine dentin powder, then completely demineralized with 10% H₃PO₄ solution. The control group was untreated, while the four experimental groups were treated by plasma jet for 20 s, 30 s, 40 s and 50 s. The crosslinking degree of each group was determined by ninhydrin colorimetric method. (3) Forty intact third molars were collected to obtain dentin strips. Only two central symmetrical dentin strips (nasty 80) were taken from each tooth and immersed in 10% H₃PO₄ solution for 16 h to obtain fully demineralized dentin collagen. Eighty dentine collagen fiber strips were randomly divided into five groups. The control group was untreated and the axial surfaces of dentin collagen fiber strips in the expe-rimental groups were treated with the plasma jet for 20 s, 30 s, 40 s and 50 s. The ultimate tensile strength of dentin was measured by universal mechanical machine. RESULTS: (1) The surface morphology of demineralized dentin observed by scanning electron microscope showed that the network structure of collagen fibers on the surface of demineralized dentin treated with the plasma jet for 20 s, 30 s and 40 s could maintain the uncollapsed three-dimensional structure, and part of the microstructure was destroyed after plasma jet treated for 50 s. After being treated by plasma jet for 20 s, 30 s and 40 s, the three-dimensional structure was uncollapsed, and the typical periodic transverse pattern of natural type Ⅰ collagen fiber could be seen. The results of infrared spectra showed that the secondary conformation of dentin collagen fibers was the same as that of the control group after being treated with the plasma jet, and the intensity of amide band was significantly increased after being treated for 30 s and 40 s. (2) The results of ninhydrin crosslinking test showed that the crosslinking ratio of dentin collagen treated by plasma jet for 30 s and 40 s was the highest, and the difference was statistically significant (P < 0.05). (3) The results of dentin ultimate tensile strength showed that the control group was (1.67±0.24) MPa, and the plasma jet treated 20 s, 30 s, 40 s and 50 s groups were (4.21±0.15) MPa, (7.06±0.30) MPa, (7.32±0.27) MPa, and (6.87±0.17) MPa, which were significantly different from that of the control group (P < 0.05). CONCLUSION The novel RF-APGD plasma jet treatment can promote the crosslinking degree of demineralized dentin collagen and improve their ultimate tensile strength.
Collagen ; Dental Bonding ; Dentin ; Dentin-Bonding Agents ; Microscopy, Electron, Scanning ; Tensile Strength

OBJECTIVE: To construct an acute myeloid leukemia cell line stably expressing CD123-CLL1 so as to provide an "in vitro" model for studying the role of CD123 and CLL-1 in leukemia and the treatment targeting CD123 and CLL-1. METHODS: The recombinant plasmid of lentivirus was constructed by synthesizing CD123 and CLL-1 sequences and PCR homologous recombination. The lentivirus vector was packaged by three-plasmid packaging system. After collecting the supernatant of lentivirus, the virus titer was determined by quantitative PCR. K562 leukemia cells were collected and transtected with virus supernatant. Leukemia cell line stably expressing the target gene were screened by purinomycin. The expression levels of CD123 and CLL-1 were detected by RT-PCR and flow cytometry. RESULTS: The lentiviral vector was successfully constructed, and identified by agarose gel electrophoresis and gene sequencing, then the virus titer of the supernatant was up to 5.81×10 CONCLUSION Lentiviral vector expressing CD123-CLL1 has been successfully constructed, and K562 leukemia cell line stably expressing CD123 and CLL-1 has been successfully obtained.
Cell Line, Tumor ; Genetic Vectors ; Humans ; Interleukin-3 Receptor alpha Subunit ; K562 Cells ; Lentivirus/genetics* ; Leukemia, Lymphocytic, Chronic, B-Cell/genetics* ; Plasmids ; Transfection

The application of special acupoints for different primary symptoms of chronic gastritis in ancient literature of acupuncture and moxibustion was analyzed and summarized. With keywords, electronic and manual retrieval of ancient literature being performed to establish a database, the association rules were performed with SPSS Modeler 18. As a result, ① among all the 2243 items included, 109 items mentioned special acupoints (excluding those only mentioned crossing acupoint). The frequency of special acupoints was 2554 (71.7%), and the special acupoints on the spleen meridian, the stomach meridian, the conception vessel were frequently used. The frequency of five- point was highest, followed by front- points. ② The special acupoints for four main primary symptoms of chronic gastritis, "stomachache" "vomiting and regurgitation" "belching and acid regurgitation" and "epigastric fullness", included Zusanli (ST 36), Neiguan (PC 6), Zhongwan (CV 12), Gongsun (SP 4), Taibai (SP 3). In addition, the back- points were also selected to treat the primary symptoms other than "epigastric fullness". Zhangmen (LR 13) was added to treat "belching and acid regurgitation". The combination of Neiguan (PC 6) and Gongsun (SP 4) showed the strongest correlation; due to different primary symptoms, the combination had different emphasis.

OBJECTIVE: To investigate the effect of different treatment temperatures of a novel cold atmospheric plasma jet treatment on the resin-dentin bonding. METHODS: (1) Fifty-two freshly extracted, non-carious and intact third molars were collected. The occlusal one-third of the crown was removed by means of a water-cooled low-speed Isomet saw. One dentin disc [(900 ±100) μm] was prepared for each tooth. The fifty-two dentin discs were randomly divided into control group and experimental groups, of which four were in control group, and forty-eight were divided into four experimental groups according to the different treatment temperatures (4 °C, 10 °C, 20 °C and 30 °C) of the novel radio-frequency atmospheric-pressure glow discharge (RF-APGD) plasma jet, twelve in each group. Each experimental group was divided into three subgroups according to different treatment time (10 s, 20 s and 30 s), with four in each subgroup. The occlusal one-third of the crown was removed by means of a water-cooled low-speed Isomet saw. The morphology of demineralized dentin surfaces was analyzed using field emission scanning electron microscopy. (2) Twenty unerupted, non-carious and intact third molars were randomly divided into five groups, four in each group: control group, untreated; 4 °C, 10 °C, 20 °C and 30 °C experimental groups, each group was treated with the RF-APGD plasma jet for 20 s. The micro-tensile resin dentin bond strength was tested after 20 s RF-APGD plasma jet treatment with different temperatures, using a universal mechanical machine. RESULTS: (1) The field emission scanning electron microscopy results indicated that when compared with the control group, a 10 s RF-APGD plasma jet treatment with 30 °C and 20 °C collapsed the collagen scaffold. Collagen fibrils maintained an uncollapsed three-dimensional structure after the 4 °C RF-APGD plasma jet treatment for even 30 s treatment. (2) The microtensile resin dentin bond strength results of the 4 °C RF-APGD plasma jet treatment group (57.8±0.7) MPa were significantly higher than that of the control group [(47.4±0.5) MPa] and 10 s, 20 s and 30 s RF-APGD plasma treatment group [(51.9±0.7) MPa,(29.7±1.0) MPa and (22.2±1.5) MPa] with statistically significant difference (P<0.05). Compared with the control group, the micro-tensile bond strength increased about 21.9% and 9.5% after 4 °C and 10 °C RF-APGD plasma jet treatment, respectively. CONCLUSION Compared with other treatment temperatures, this novel RF-APGD plasma jet treatment with the temperature of 4 °C can preserve the three-dimensional morphology of demineralized dentin better, and can improve the resin-dentin bonding.
Composite Resins ; Dental Bonding ; Dentin ; Dentin-Bonding Agents ; Materials Testing ; Microscopy, Electron, Scanning ; Plasma Gases ; Resin Cements ; Temperature ; Tensile Strength

BACKGROUND: Inducing factors are currently used as a main method for the differentiation of bone marrow mesenchymal stem cells (BMSCs) into chondrocytes. OBJECTIVE: To investigate the collaborative stimulation of transforming growth factor beta1 (TGF-β1) and insulin-like growth factor 1 (IGF-1) to induce the directed differentiation of BMSCs to chondrocytes, and to explore the best inductive effect. METHODS: Rat BMSCs were isolated, cultured and purified using adherent culture. Then, different inducing factors were added in the induction medium: TGF-β1+IGF-1 group, TGF-β1 group, IGF-1 group, and control group without growth factors. Immunofluorescence was carried out at 21 days of induction. The expression of collagen type Ⅱ was evaluated by immuncytochemical staining at 7, 14 and 21 days of induction. RESULTS AND CONCLUSION: (1) Immunofluorescence detection of the TGF-β1+IGF-1 and TGF-β1 groups showed highly expressed collagen type Ⅱ (brown red-stained cytoplasm), while negatively expressed collagen type Ⅱ in the other two groups. (2) Findings from the immuncytochemical staining showed that the expression of collagen type Ⅱ was stronger in the TGF-β1+IGF-1 group than the TGF-β1 group (P < 0.01), and the expression was gradually enhanced with time. Meanwhile, there was also no expression of collagen type Ⅱ in the IGF-1 and control groups. To conclude, the combination of TGF-β1 and IGF-1 can achieve the better inductive effect on the chondrogenic differentiation of BMSCs in vitro.