Traditional development of small protein scaffolds has relied on display technologies and mutation-based engineering, which limit sequence and functional diversity, thereby constraining their therapeutic and application potential. Protein design tools have significantly advanced the creation of novel protein sequences, structures, and functions. However, further improvements in design strategies are still needed to more efficiently optimize the functional performance of protein-based drugs and enhance their druggability. Here, we extended an evolution-based design protocol to create a novel minibinder, BindHer, against the human epidermal growth factor receptor 2 (HER2). It not only exhibits super stability and binding selectivity but also demonstrates remarkable properties in tissue specificity. Radiolabeling experiments with ^99mTc, ⁶⁸Ga, and ¹⁸F revealed that BindHer efficiently targets tumors in HER2-positive breast cancer mouse models, with minimal nonspecific liver absorption, outperforming scaffolds designed through traditional engineering. These findings highlight a new rational approach to automated protein design, offering significant potential for large-scale applications in therapeutic mini-protein development.

To assess and grade facial nerve dysfunction according to the extent of facial paralysis in the clinical course of acupuncture treatment for Bell's palsy, and to observe the interrelationship between the grade, the efficacy and the period of treatment, as well as the effect on prognosis.

ObjectiveTo investigate the effects of imatinib mesylate as a tyrosine kinase inhibitor on the biological activity of and Wnt/β-catenin pathway in Hs294T melanoma cells.MethodsAfter Hs294T cells were incubated with imatinib mesylate at various concentrations(4,8,10,16,20 and 24 μmol/L) for 24 hours or imatinib mesylate at 10 μmol/L for 24,48 and 72 hours,methyl thiazolyl tetrazolium (MTT) assay was performed to estimate the proliferation of cells and to determine the effects of imatinib mesylate on the proliferation of Hs294T cells.Then,Hs294T cells were treated with imatinib mesylate at 10 μmol/L or dimethyl sulfoxide (DMSO) for different durations,followed by the detection of cell apoptosis with flow cytometry,localization of β-catenin with annexin V/propidium iodide-double staining and laser confocal microscopy,quantification of β-catenin and cyclin D1 protein with Western blot,and measurement of LEF1 and C-myc mRNA expression with real time fluorescence-based quantitative PCR.Matrigel invasion assay was performed to evaluate the invasiveness of Hs294T cells after treatment with imatinib mesylate at 5 μmol/L or DMSO for 24 hours.ResultsImatinib mesylate at 4-10 μmol/L elicited a dose-dependent decline in the proliferation of Hs294T cells (F =125.3,P ＜ 0.05),and imatinib mesylate at 10 μmol/L induced a time-dependent decrease from 24 to 72 hours(F =714.6,P ＜ 0.01 ).The percentage of early and late apoptotic cells was markedly increased,while the invasiveness was decreased by about 48％(P ＜ 0.01 ),together with a downregulation in the expression of LEF1,C-myc and Cyclin D1 in imatinib mesylate-treated Hs294T cells compared with the DMSO-treated cells.No obvious changes were observed in the protein expression of β-catenin,but a decline in the nuclear localization of β-catenin was noted in Hs294T cells after being treated with imatinib mesylate.ConclusionImatinib mesylate may suppress the proliferation and invasion of,but promote the apoptosis in,melanoma cells,by downregulating the Wnt/β-catenin pathway.

Objective To investigate the in vitro effects of KAAD-cyclopamine, a specific inhibitor of hedgehog signaling pathway, on the growth and apoptosis of human squamous cell carcinoma cell line A431. Methods A431 cells were cultured and treated with KAAD-cyclopamine(0.5, 1, 2, 5 μmol/L).Then, MTT assay was used to detect the proliferation of A431 cells, and light microscopy to observe cell morphology at different time points with a 24-hour interval. Flow cytometry was used to assess cell cycle,and annexin-V/propidium iodide double staining to evaluate the apoptosis in these cells after 48 hours of treatment with KAAD-cyclopamine. Results KAAD-cyclopamine of 0.5, 1, 2 and 5 μmol/L inhibited the proliferation of A431 cells by (7.0±2.3)%, (20.6±2.8)%, (48.3±3.4)% and (61.6±3.3)%, respectively (F = 49.92, P＜0.01 ). Furthermore, in the presence of KAAD-cyclopamine of 5 μmol/L, on day 1, 2, 3, 4,and 5 the proliferation of A431 cells was suppressed by (18.5±2.6)%, (56.1±3.7)%, (65.4±2.8)%,(71.2±1.9)% and (75.9±3.0)%, respectively, the difference was significant among these time points(F =16.32, P＜0.01 ). Statistical analysis showed that KAAD-cyclopamine downregulated the growth of A431 cells in a dose-and time-dependent manner (r = 0.91, 0.86, P＜0.01 and 0.05, respectively). Light microscopy revealed typical morphological changes of cell damage in A431 cells. KAAD-cyclopamine in creased the percentage of cells in G1 phase from (51.8±2.9)% to(76.2±1.8)% (F = 26.34, P＜0.01 ), the proportion of hypoploid cells from (1.7±0.3 )% to (8.7±0.2)% (F = 6.32, P＜0.05 ), which suggested that KAAD-cyclopamine could arrest A431 cells in G1 phase of the cell cycle. The apoptosis ratio in KAAD-cyclopamine-treated cells was significantly higher than that in the untreated control [ (46.2±2.8)% vs (18.5±3.1 )%, F = 32.01, P＜0.01 ]. Tomatidine treatment did not affect the proliferation or apoptosis of A431 cells (both P＞0.05).Conclusion KAAD-cyclopamine can markedly suppress the proliferation and induce apoptosis of A431 cells.

BACKGROUND: Apatite-wollastonite containing glass-ceramic (AWGC) is a kind of good bone repairing materials with excellent bioactivity, which is prepared by traditional melting process.OBJECTIVE: To observe AWGC prepared with sol-gel method and its bioactivity.DESIGN: Design experiment of materials process and in vitro bioactivity experiment.SETTING: College of materials science and Engineering of Sichuan University.MATERIALS: AWGC.METHODS: This experiment was conducted at the laboratory of College of Materials Science and Engineering of Sichuan University between August 2002 and May 2003. AWGC was prepared from sol-gel and followed by heattreating process. Bioactivity was investigated in vitro by immersing in the simulate body fluid (SBF) at 37 ℃ for 7 days . JL-1155 laser particle analyzer, X-ray diffraction, Fourier transform infrared spectroscopy and scanning electron microscope were used for micro-morphological structure analysis.MAIN OUTCOME MEASURES: ①The crystalline structure and microstructure of sol-gel derived glass-ceramic② The apatite forming process in simulate body fluid③ The diameter of the pore of the sol-gel derived apatite/wollastonite glass-ceramicRESULTS: ①Main crystalline phases of the sol-gel derived glass-ceramic materials were hydroxyapatite/fluoroapatite [Ca10(PO4)6(OH, F)] and β-wollastonite[β-CaSiO3]; Microstructure contained many micro-pores of 2-3μ m;② Sol-gel derived AW glass ceramic had excellent bioactivity: plenty of apatite granules were generated on the surface of the material after soaking for 7 days. ③Porous scaffolds possessed good macro-porous structure with the interconnected macro pores of 300-400 μm in diameter;CONCLUSION: Apatite-wollastonite containing glass-ceramic (AWGC)with excellent bioactivity was developed by sol-gel process. The material is expected to be a good candidate for bone-repairing and bone tissue engineering scaffold materials.