A novel amplifier system was proposed for low-noise recording of pico-ampere current in nanopore experiment (<100 pA). As an example, the amplifier system was applied in α-hemolysin based nanopore detection of DNA-PEG-DNA conjugate to record the signals of translocation and bumping events in buffer solution (1 mol/L KCl, 10 mmol/L Tris--HCl, 1 mmol/L EDTA and pH=8. 0). The amplified current signal was filtered by a 3 kHz Bessel filter and sampled by a 100 kHz analog-digital convertor. As a result, the presented amplifier system could lower the noise in recording the current. The current blockages (<10 pA) of single molecules with low amplitude were recovered due to the high signal-to-noise ratio.

Objective:To explore a modified flap bridging procedure in primary free flap transfer to reconstruct defects in calf and with secondary reconstruction of the great saphenous vein of the calf according to the diagnoses of 3 types of traumatic calf ulcers, and to observe the clinical effect.Methods:A research on evidence-based medicine was applied from April 2010 to June 2023 in the Department of Orthopaedics of the Second People's Hospital of Hefei on 25 patients with calf traumatic ulcers. The traumatic calf ulcers were classified into 3 types: Type I, a defect type (5 patients), Type II, a scar type (8 patients), and Type Ⅲ, a osteomyelitis type (12 patients). The age of patients ranged from 22 to 67 years old, with an average of 44 years old. The course of calf traumatic ulcers from 3 months to 36 years, with 17 in left calf and 8 in the right. The preoperative Lower Extremity Function Scale (LEFS) was 37.07 points±18.92 points. The modified flap bridge surgery was planned in 2 stages. The first stage surgery was simultaneously performed by 2 surgical teams, with surgical team 1 carried out ulcer debridement and preparation for a flap bridging surgery. According to the 3 types of diagnosis, the infected and unhealthy tissues were completely removed by individualised debridement, the sizes of defect were 5 cm×11 cm-14 cm×27 cm. Medial flaps were dissected to prepare a flap bridging (including great saphenous vein) with the proximal calf of the healthy side as the rotation point. Both lower legs were then fixed together. For an unhealed Type I or III fracture, the tibial fracture was reduced and fixed with an external fixator and then fixed with the contralateral tibia. For a healed Type II or III fracture, both calcaneus were fixed together with a Steinmann pin in obliquely penetration. The surgical team 2 carried out the task of harvest of a free flap. The flap with both adjustable design and anatomy was selected. The flap donor sites with a larger area and dominant vein were the best: anterolateral thigh flap (13 patients), thoracoumbilical flap (9 patients) and lateral thoracic flap (3 patients). The sizes of the transferred flaps were of 6 cm×12 cm-15 cm×28 cm. Eighteen patients had direct suture of donor sites and 7 were reconstructed with medium-thick skin grafts. The 2 teams then worked together: to cover the defect with the flap, anastomose the artery carried by flap with the posterior tibial artery of the bridge, flap dominant vein with the great saphenous vein of the bridge and flap, and non-dominant vein with the dominant posterior tibial vein of the bridge respectively, and then suture the wounds. Stage II surgery: it was carried out at 5-6 weeks later. In which, pedicle of the flap bridge was disconnected, and the great saphenous vein (including 3-4 venous valves) with a ≥10 cm in length, was dissected to the proximal end of the flap bridge. The end of it was anastomosed with the great saphenous vein proximal to the end of the affected limb. The flap bridge and the vascular bundle behind the tibia were restored and replanted. Postoperative wound management and follow-up were carried out and LEFS were collected.Results:All 25 patients were included in the postoperative follow-up for 6-18 (average 10.5) months. CDU showed that the reconstructed great saphenous veins were unobstructed. All the ulcers were cured and the appearances of the calfs were satisfactory with good texture, and without recurrence of ulcers. There was a linear scar in donor sites and without scar formation at the sites of skin graft and skin extraction. The last LEFS was 67.32 points±8.43 points. All patients returned to normal life and work.Conclusion:Classification of traumatic calf ulcer into 3 types has a theoretical basis in selection and modification of flap bridging surgery. The surgery has characteristics of individuation and modularisation, it not only benefits the reconstruction of ulcerative defect, but also prevents the recurrence of ulcer. It is a safe and effective surgical procedure with clinical proof.

[Abstract] Objective:To prepare the third generation CAR-T cells targeting EGFRvⅢ (EGFRvⅢCAR-T) and to detect its specific killing effect against EGFRvⅢ+ U87 cells in vitro and in vivo. Methods: Human CD3+ T cells were transfected with lentiviral EGFRv Ⅲ/3CAR, which was generated by calcium phosphate co-precipitation of three plasmids. The expression of EGFRvⅢ/3CAR in T cells was detected by Western blotting and flow cytometry. In vitro killing effect of EGFRvⅢ/3CAR-T cells on EGFRvⅢ+ U87 cells was detected by 51Cr release assay. The secretion of cytokine IFN-γ of EGFRvⅢ/3CAR-T cells was detected by ELISA. Nude mouse xenograft model was constructed to detect the in vivo cytotoxicity of EGFRvⅢ/3CAR-T cells on xenograft tumor. Results: The EGFRvⅢ/3CAR lentivirus was successfully packaged with an average titer of 5×106 TU/ml. Western blotting showed that a protein band of approximate 58 000 molecular weight was observed in EGFRvⅢ/3CAR-T cells but absent in untransfected T cells. Flow cytometry indicated the average transduction efficiency of EGFRvⅢ/3CAR was 52.3%. 51Cr release assay showed that the specific killing effect of EGFRvⅢ/ 3CAR-T cells was positively correlated with E/T ratio (E∶T=4∶1, 8∶1, 16∶1, 32∶1). ELISA showed that cytokine IFN-γ secretion was (1 836±148.2) pg/ml, which was significantly different from that of NTT and GFP+ T cells (P<0.01). The specific killing activity of EGFRvⅢ/3CAR-T cells and IFN-γ secretion were both dependent on the expression level of EGFRvⅢ in U87 cells. The tumor growth monitoring results showed that the tumor volume of EGFRvⅢ/3CAR-T cell group was significantly different from that of GFP+ T cell group and PBS group around 3 weeks after injection (P<0.01). Conclusion: EGFRvⅢ/3CAR-T cells demonstrated specific antitumor effectagainstEGFRvⅢ+U87cellsbothinvitro and in vivo, providing basis for immunotherapyofgliomainfuture clinical use.

Objective: To explore the gene transduction method of chimeric antigen receptor (CAR) mediated by novel cationic polymer nanocarrier mPEG-P (Asp-AED-g-HFB) (PAEF) and PigyBac transposon system to modify natural killer (NK) cells, providing a new strategy for immunotherapy of cancer cells. Methods: PAEF/DNA (transposase+transposon) complex were prepared. The particle size distribution and surface potential of PAEF/DNA complexes were measured with Nano-ZSE Dynamic Light Scattering System (Malvern Instruments). The DNA encapsulation rate, release and stability of PAEF were evaluated by DNA gel electrophoresis, and then by combiningwithparticlesizeandsurfacepotentialtodeterminethepreferentialN/PratiotoenterNKcells.Thecell cytotoxicity of PAEF/DNA complexes under different N/P ratios was analyzed by CCK-8 cytotoxicity test. Transduction efficiency of NK cells was evaluated by Fluorescence microscopy and Flow cytometry, and the feasibility of PAEF gene transfection vectors was assessed. Results: PAEF could encapsulate DNA to form nano-complexes with the diameter of 100-150 nm, which was suitable to mediate DNA entering into cells. PAEF could completely encapsulate DNA with N/P ratio of 20. In the presence of reducing agent dithiothreitol (DTT), PAEF had a good ability to release DNA. NK-92 cells transfected with PAEF/DNA complex, which was formed at the N/P ratio of 80, attained a significantly higher cell viability than cells of lipofectamine transfection group [(72.50±3.9)% vs (64.03±1.8)%, P<0.05]; Fluorescence microscopic observation showed more fluorescence and higher fluorescence intensity in cells of PAEF/DNA group; Flow cytometry showed the highest transfection efficiency of 83.4%. Conclusions: Nanocarrier PAEF can encapsulate DNA well by electrostatic adsorption, and has good biocompatibility and high efficiency for gene transduction. It provides a good experimental basis for adoptive immunotherapy.