High performance production process development and scale-up of an anti-TSLP nanobody.

Nanobodies (Nbs) represent a class of single-domain antibodies with great potential application value across diverse biotechnology fields, including therapy and diagnostics. Thymic Stromal Lymphopoietin (TSLP) is an epithelial cell-derived cytokine, playing a crucial role in the regulation of type 2 immune responses at barrier surfaces such as skin and the respiratory/gastrointestinal tract. In this study, a method for the expression and purification of anti-TSLP nanobody (Nb3341) was established at 7 L scale and subsequently scaled up to 100 L scale. Key parameters, including induction temperature, methanol feed and induction pH were identified as key factors by Plackett-Burman design (PBD) and were optimized in 7 L bioreactor, yielding optimal values of 24 °C, 8.5 mL/L/h and 6.5, respectively. Furthermore, Diamond Mix-A and Diamond MMC were demonstrated to be the optimal capture and polishing resins. The expression and purification process of Nb3341 at 100L scale resulted in 22.97 g/L titer, 98.7% SEC-HPLC purity, 95.7% AEX-HPLC purity, 4 ppm of HCP content and 1 pg/mg of HCD residue. The parameters of the scaling-up process were consistent with the results of the optimized process, further demonstrating the feasibility and stability of this method. This study provides a highly promising and competitive approach for transitioning from laboratory-scale to commercial production-scale of nanobodies.

MicroRNA-346-5p Regulates Differentiation of Bone Marrow-Derived Mesenchymal Stem Cells by Inhibiting Transmembrane Protein 9.

The molecular mechanisms how bone marrow-derived mesenchymal stem cells (BMSCs) differentiate into osteoblast need to be investigated. MicroRNAs (miRNAs) contribute to the osteogenic differentiation of BMSCs. However, the effect of miR-346-5p on osteogenic differentiation of BMSCs is not clear. This study is aimed at elucidating the underlying mechanism by which miR-346-5p regulates osteogenic differentiation of human BMSCs. Results of alkaline phosphatase (ALP) and Alizarin Red S (ARS) staining indicated that upregulation of miR-346-5p suppressed osteogenic differentiation of BMSCs, whereas downregulation of miR-346-5p enhanced this process. The protein levels of the osteoblastic markers Osterix and Runt-related transcription factor 2 (Runx2) were decreased in cells treated with miR-346-5p mimic at day 7 and day 14 after being differentiated. By contrast, downregulation of miR-346-5p elevated the protein levels of Osterix and Runx2. Moreover, a dual-luciferase reporter assay revealed that Transmembrane Protein 9 (TMEM9) was a target of miR-346-5p. In addition, the Western Blot results demonstrated that the TMEM9 protein level was significantly reduced by the miR-346-5p mimic whereas downregulation of miR-346-5p improved the protein level of TMEM9. These results together demonstrated that miR-346-5p served a key role in BMSC osteogenic differentiation of through targeting TMEM9, which may provide a novel target for clinical treatments of bone injury.

Anti-tumor effects of dihydroartemisinin on human osteosarcoma.

Dihydroartemisinin (DHA) exhibits antitumor activity against a wide spectrum of cancer cells. However, whether DHA has anti-tumor effect on human osteosarcoma cells remains unknown. This study aims to investigate the anti-tumor activity of DHA and the underlying mechanisms in human osteosarcoma cell lines with different p53 mutation statuses. Four human osteosarcoma cell lines were treated with different concentrations of DHA. Then, cell proliferation was determined by the CCK-8 viability assay; apoptosis and cell cycle progression were evaluated by flow cytometry; protein expression was analyzed by western blot assay; and NF-kB activity was examined by luciferase assay. The results demonstrated that DHA treatment could inhibit the proliferation of four osteosarcoma cell lines in a dose-dependent manner. P53 wild-type osteosarcoma cells were more sensitive to DHA. Moreover, the percentage of apoptotic cell and cell arrest in G2/M phase was increased upon DHA treatment in a dose-dependent manner. Mechanistically, DHA activated caspase-3, caspase-8, and caspase-9; upregulated the expression of Bax, FAS, and cyclin D1; downregulated the expression of Bcl-2, Cdc25B, and cyclin B1; and inhibited the activity of NF-кB. In conclusion, DHA has significant anticancer effects against human osteosarcoma cells, which include induction of apoptosis and cell cycle arrest. The p53 gene may play a certain role in the DHA-induced human osteosarcoma apoptosis and cell cycle arrest. DHA is a novel anti-osteosarcoma drug candidate that merits further study.