Accurate simulation is the key of improve local control rates for radiotherapy esophageal carcinoma. CT simulation is an excellent tool that advances our capabilities of 3-D conformal radiation therapy so that higher radiation dose can be delivered to the target with a corresponding expectation of increased tumor control, possibly combined with a decrease in morbidity for radiotherapy esophageal carcinoma. PET/CT has an advantage in delineation of lateral extension of esophageal tumor, has more values for simulation in radiotherapy esophageal.

[Objective]To explore the potential of a novel collagen I /chitosan /Nano?-tricalcium phosphate(?-TCP)bilayered scaffold for being used in tissue engineering(TE)of osteochondral repair.[Method]Bilayered scaffolds were produced with collagen Ⅰ,chitosan and?-TCP,using a special cross-linking and freeze drying method.The pore size,porosity and interpores of the scaffold were observed by scanning electron microscopy(SEM).Rabbit bone mesenchymal stem cells(BMSC)were isolated and amplified,then inoculated onto the scaffold.By SEM scanning,the condition of the cells adhering onto the scaffold was observed.The proliferation of the cells on the scaffolds was examined using MTT method,and the growth curve was drawn.The cell-scaffold composite were then induced to differentiate towards cartilage by 3-D culturing,and then implanted into muscle pouches 2 weeks later.The result was observed 6 weeks later by HE staining,toluidine blue staining and type Ⅱ collagen immunohistochemistry.[Result]The scaffold possessed high porosity and proper pore size,the porosity was above 95%.BMSC could adhere onto the scaffold well,and the proliferation rate of the cells on the scaffolds was perfectly good.After in vitro induction,BMSC-scaffold composite can differentiate toward cartilage ectopicly.[Conclusion]The novel collagen I /chitosan /?-TCP bilayered scaffold possesses good pore structure and biocompatibility,and will possibly become a new biomaterial of TE used for osteochondral repair.

Objective To investigate the setup errors of super chest segment of esophageal cancer patients before radiotherapy delivery by KV cone beam CT,and evaluate the margin from CTV to PTV.Methods From 2010 to 2012,13 patients with super chest segment of esophageal cancer whose IMRT planning CT images were included in this study.Delineate target on the CT images of treatment planning and enlarge the margin of CTV to form ITV,then enlarge the margin of ITV gradually 10 times by 1 mm each time to form varied PTV,and create the plan according to the size of the PTV,simulate setup errors in the new plan to obtain the simulation of the actual exposure curve and find a suitable PTV to assure 95％ ITV volume as ever to approach the prescription dose,obtained the outside enlarge distance of CTV → PTV.Results The maximum setup errors in the direction of the anterior and posterior positioning was (3.42 ±2.19) mm.The margin of ITV→PTV is 5 mm which was figured out by PTN enlarging method.Compared to the original plan that under the condition of draw up the radiotherapy plan that based on the method of PTV enlarging obtained the CTV→PTV and simulate the actual dose distribution according to the setup errors:total lung V5,spinal cord D1cm3,increased by about 0.87％,4.95 Gy,heart V40,PTV D95,PTV V100,ITV D95,ITV V100 were reduced about 0.62％,4.95 Gy,8.38％,1.84 Gy,1.87％,all of them have statistically difference.Conclusions Range of external expansion of the left to right,superior to inferior and anterior to posterior is 7 mm,8 mm and 7 mm respectively,according to the method of PTV enlarging obtained the margin of CTV→PTV of super chest segment of esophageal cancer patients.

BACKGROUND: Whether the injured peripheral nerve in a late stage has repairing value still remains a problem. If irreversible changes happen in substance P and calcitonin gene-related peptide, the feeling function will lose even after repairing.OBJECTIVE: To quantitatively study the changes of substance P(SP) and calcitonin gene-related peptide(CGRP) in the spinal dorsal horn 24 weeks after peripheral nerve injury.DESIGN: A self-controlled quantitative experiment.SETTING: Institute of Orthopaedics of the Fourth Military Medical University of Chinese PLA.MATERIALS: This experiment was performed in the Institute of Orthopaedics of the Fourth Military Medical University of Chinese PLA from October 2002 to May 2003. Totally 55 SD rats were divided into 11 groups according to time points(1, 2, 3, 4, 6, 8, 10, 12, 16, 20 and 24 weeks after sciatic nerve transection).INTERVENTIONS: Sciatic nerve injury model was set up by transecting one side of sciatic nerve and ligating the proximal stump of sciatic nerve; the other side was set as the control side. Computer-assisted image analysis was used to measure the immunologic reaction areas of substance P and CGRP.MAIN OUTCOME MEASURES: Changes of the distribution of the positive fibres of SP and CGRP in rat spinal dorsal horn in each group.RESULTS: Fifty-five rats entered the result analysis. The distributions of SP immunoreactivity in the spinal dorsal horn following sciatic nerve injury showed a significant reduction during 2-6 weeks, followed by a slow rate of increase,and reached almost complete restoration at 16 weeks after deafferentation. No obvious advanced changes happened at 20 and 24 weeks. The ratios for ipsilateral and cotralateral sides of positive fibre and distribution area injury in spinal dorsal horn CGRP were 1.14 at week 1, 1.13 at week 6, and 0. 29 at week 24. The ratios were similar at each time point( P ＞ 0. 05).CONCLUSION: At the late stage of peripheral nerve injury, neurons in the spinal dorsal horn and dorsal root ganglion still remain their functions to synthesize and secrete SP and CGRP. Spinal dorsal horn remains at a balanced status and still has the neurologic basis to recover the sensory function.

Objective To investigate the regulatory effects of miR-146a on inflammation factors production in C ryptococcus neoformans treated THP-1 cells.Methods Cryptcoo ccus neoformans strains were heat killed .Fluorescence quantitative RTP-CR and ELISA were used to detect the levels of IL -1βand TNF-αin THP-1 cells treated with heat-killed Cryptococcus neoformans.In addition, the production of IL-1βand TNF-αwere analyzed before and after Dectin-1or TLR 4 blocked .THP-1 cell lines that were respectively transfected with miR-146 a mimics and inhibitors were constructed and the production of IL-1βand TNF-αin these cell lines were analyzed after interfered with Cryptococcus neoformans.Results With the interference of heat-killed Cryptococcus neoformans, the expression of miR-146a was up-regulated in THP-1 cells, but was down-regulated after Dectin-1 or TLR4 blocked.The expressions of IL-1βand TNF-αinduced by heat-killed Cryptococcus neoformans were enhanced in miR-146a mimics transfected THP-1 cells, but was inhibi-ted in inhibitors transfected THP-1 cells.Conclusion Heat-killed Cryptococcus neoformans could up-regu-late miR-146a expression in THP-1 cells via Dectin-1 and TLR.miR-146a could negatively regulate the ex-pressions of IL-1βand TNF-αinduced by Cryptococcus neoformans.

Tri-dimensional poly (DL-lactic-co-glycolic acid) (PLGA) scaffolds were fabricated using a rapid prototyping (RP) technique and the gene of human bone morphogenetic protein 2 (hBMP-2) was transferred into rabbit bone marrow stromal cells (MSCs) via recombinant adeno-associated virus vectors (rAAV-hBMP-2). Thirty-two PLGA scaffolds, size (4 mm X 4 mm X 4 mm), were coated with collagen type I and equally divided into 2 groups. In group A, each scaffold was loaded with 2 X 10(4) hBMP-2 (+) MSCs to establish a hBMP-2 (+) MSCs/PLGA composite. In group B, each scaffold was loaded with 2 X 10(4) hBMP-2 (-) MSCs to establish a hBMP-2 (-) MSCs/PLGA composite. The composites in both groups were cultured for subcutaneous implantation in nude mice. All animals were killed 30 days after implantation and the differentiation of composites was evaluated. As a result, MSCs infected with rAAV-hBMP-2 efficiently expressed hBMP-2 protein. RP-based PLGA scaffolds had ideal microarchitecture. The diameters of macropore and micropore of the scaffolds were 300 microm and 3-5 microm, respectively. At 3-5 days after culture, a number of seeding cells well grew on the scaffolds of both groups. The composites in group A had chondrogenesis ability in vivo and the expression of collagen type II was positive. In group B, however, only polymers and fiber tissues were predominantly found. The percentage of polymer remnant area was significantly lower in group A than in group B (P<0.01). Our results therefore indicate that RP-based PLGA scaffolds efficiently coated with collagen type I have good biocompatibility with hBMP-2 (+) MSCs and the techniques developed in this study may favor cartilage tissue engineering.
Animals ; Biocompatible Materials ; Bone Marrow Cells ; cytology ; Bone Morphogenetic Protein 2 ; Bone Morphogenetic Proteins ; genetics ; Cell Differentiation ; Cells, Cultured ; Chondrogenesis ; Guided Tissue Regeneration ; methods ; Humans ; Implants, Experimental ; Lactic Acid ; Male ; Mice ; Mice, Nude ; Polyglycolic Acid ; Polymers ; Rabbits ; Stromal Cells ; cytology ; Tissue Engineering ; methods ; Transfection ; Transforming Growth Factor beta ; genetics

Under laboratory condition, the compound materials of Poly (DL-lactic-co-glycolic acid)/Tricalcium phosphate [PLGA/TCP(L), with component ratio of 7:3] were fabricated by combining the thermally induced phase separation (TIPS) with solvent-casting particulate-leaching (SCPL) approach. On the other hand, rapid prototyping (RP) technique manufactured PLGA/TCP scaffolds [PLGA/TCP(RP)] were obtained. These two kinds of carriers were coated with collagen type I (Col I). The extracted bovine bone morphogenetic protein (bBMP) was loaded into carriers to establish biomimetic synthetic bones. PLGA/TCP(L) scaffolds, demineralized bone matrices (DBM) of bovine cancellous bone, PLGA/TCP(L) scaffolds, biomimetic synthetic bones and OsteoSet bone graft substitutes were investigated. Scanning electron microscopy revealed that the microarchitecture of PLGA/TCP(RP) scaffolds was much better than that of PLGA/TCP(L) scaffolds. The diameter of macropore of PLGA/TCP(RP) scaffold was 350 microm. The porosities of PLGA/ TCP(L) scaffolds, DBM, PLGA/TCP(RP) scaffolds and OsteoSet bone graft substitutes were 21.5%, 70.4%, 58.6% and 0%, respectively (P<0.01). Modification of PLGA/TCP scaffolds with collagen type I [PLGA/TCP(L)-Col I and PLGA/TCP(RP)-Col I] essentially increased the affinity of the carriers to bBMP. Among these synthetic materials, PLGA/TCP(RP)-Col I-bBMP composite is promising as a novel bone graft substitute due to its advanced fabrication technique, good tri-dimensional microarchitecture and ideal components.
Biocompatible Materials ; chemistry ; Bone Morphogenetic Proteins ; chemistry ; Bone Substitutes ; chemistry ; Calcium Phosphates ; chemistry ; Humans ; Lactic Acid ; chemistry ; Microscopy, Electron, Scanning ; Polyglycolic Acid ; chemistry ; Porosity ; Surface Properties ; Tissue Engineering ; methods

To develop an enzyme-linked immunosorbent assay (ELISA)-based high throughput screening (HTS) method for β-catenin/Lef1 interaction antagonists screening, Escherichia coli Rosetta (DE3) competent cells were transformed with β-catenin-pET-30a(+) plasmid. β-catenin protein was expressed after induction and purified using affinity chromatography. The biological activity of purified β-catenin was further analyzed by GST Pulldown assay. The β-catenin/GST-Lef1 binding model was established using ELISA principle, and the ELISA-based HTS method was further optimized through determination of an optimal coated concentration of GST-Lef1 and working concentration of β-catenin. The results showed that β-catenin protein was successfully expressed and purified. The GST Pulldown assay demonstrated a perfect biological activity for purified β-catenin. Subsequently, the ELISA-based HTS method was performed using 10 μg/mL GST-Lef1 and 6 μg/mL β-catenin, with the Z factor of 0.76. Taken together, we have successfully developed a simple, robust and reliable ELISA-based HTS method for screening of novel Wnt inhibitors targeting β-catenin/Lef1 interaction.
Antineoplastic Agents ; Enzyme-Linked Immunosorbent Assay ; High-Throughput Screening Assays ; Lymphoid Enhancer-Binding Factor 1 ; beta Catenin

The main protease (Mpro) of SARS-CoV-2 is a highly conserved and mutation-resistant coronaviral enzyme, which plays a pivotal role in viral replication, making it an ideal target for the development of novel broad-spectrum anti-coronaviral drugs. In this study, a codon-optimized Mpro gene was cloned into pET-21a and pET-28a expression vectors. The recombinant plasmids were transformed into E. coli Rosetta(DE3) competent cells and the expression conditions were optimized. The highly expressed recombinant proteins, Mpro and Mpro-28, were purified by HisTrapTM chelating column and its proteolytic activity was determined by a fluorescence resonance energy transfer (FRET) assay. The FRET assay showed that Mpro exhibits a desirable proteolytic activity (25 000 U/mg), with Km and kcat values of 11.68 μmol/L and 0.037/s, respectively. The specific activity of Mpro is 25 times that of Mpro-28, a fusion protein carrying a polyhistidine tag at the N and C termini, indicating additional residues at the N terminus of Mpro, but not at the C terminus, are detrimental to its proteolytic activity. The preparation of active SARS-CoV-2 Mpro through codon-optimization strategy might facilitate the development of the rapid screening assays for the discovery of broad-spectrum anti-coronaviral drugs targeting Mpro.
COVID-19 ; Codon/genetics* ; Cysteine Endopeptidases/genetics* ; Escherichia coli/genetics* ; Humans ; Peptide Hydrolases ; SARS-CoV-2 ; Viral Nonstructural Proteins/genetics*

In canonical Wnt/β-catenin signaling pathway, β-catenin/TCF4 (T-cell factor 4) interaction plays an important role in the pathogenesis and development of non-small cell lung cancer (NSCLC), and it is tightly associated with the proliferation, chemoresistance, recurrence and metastasis of NSCLC. Therefore, suppressing β-catenin/TCF4 interaction in Wnt/β-catenin signaling pathway would be a new therapeutic avenue against NSCLC metastasis. In this study, considering the principle of enzyme-linked immunosorbent assay (ELISA), an optimized high-throughput screening (HTS) assay was developed for the discovery of β-catenin/TCF4 interaction antagonists. Subsequently, this ELISA-like screening assay was performed using 2 μg/mL GST-TCF4 βBD and 0.5 μg/mL β-catenin, then a high Z' factor of 0.83 was achieved. A pilot screening of a natural product library using this ELISA-like screening assay identified plumbagin as a potential β-catenin/TCF4 interaction antagonist. Plumbagin remarkably inhibited the proliferation of A549, H1299, MCF7 and SW480 cell lines. More importantly, plumbagin significantly suppressed the β-catenin-responsive transcription in TOPFlash assay. In short, this newly developed ELISA-like screening assay will be vital for the rapid screening of novel Wnt inhibitors targeting β-catenin/TCF4 interaction, and this interaction is a potential anticancer target of plumbagin in vitro.
Carcinoma, Non-Small-Cell Lung ; Cell Line, Tumor ; Enzyme-Linked Immunosorbent Assay ; High-Throughput Screening Assays ; Humans ; Lung Neoplasms ; Transcription Factor 4/genetics* ; beta Catenin/genetics*