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Use of paclitaxel as monotherapy or in combination with other therapeutic agents is a widely employed front-line chemotherapeutic strategy for cervical cancer. However, previous reports have shown that approximately 70% of the patients with cervical cancer develop resistance to paclitaxel. Epithelial-mesenchymal transition (EMT) contributes to the occurrence of chemoresistance in several types of cancer, including cervical cancer. Identification of the critical signaling pathway that regulates the EMT process may provide a novel strategy for avoiding or delaying the emergence of paclitaxel resistance during the treatment of cervical cancer. Herein, we established a paclitaxel-resistant cervical cancer cell line (HeLa-229PTR cells) by culturing parental HeLa-229 cells with increasing concentrations of paclitaxel. We observed elevated expression of Notch1 in HeLa-229PTR cells compared with their parental HeLa-229 cells, indicating its potential involvement in the EMT phenotype of the paclitaxel-resistant cells. Furthermore, silencing of the NOTCH1 gene, as well as treatment with a γ-secretase inhibitor (DAPT) partially reversed the EMT phenotype and significantly enhanced the sensitivity of HeLa-229PTR cells to paclitaxel. Moreover, we found that DAPT could significantly inhibit invasiveness, reduce colony formation activity, and promote apoptosis of HeLa-229PTR cells. Taken together, these results indicated that HeLa-229PTR cells develop the EMT phenotype partly through activation of Notch1 signaling. Thus, inhibition of Notch1 signaling can be a strategy for the reversal of the EMT phenotype and may increase the sensitivity of cervical cancer cells to treatment with paclitaxel.

Multiple myeloma (MM) is the second most common hematopoietic malignancy and remains an incurable disease. Thus, novel drugs and therapeutic methods are required for patients with MM. The present study aimed to investigate the effect of sirtuin 1 (SIRT1) inhibitor cambinol on the proliferation and apoptosis of myeloma cell lines, RPMI8226 and U266. Moreover, the present study evaluated the underlying molecular mechanisms of proliferation inhibition and apoptosis induced by cambinol. A Cell Counting Kit-8 assay was used to measure the viability of RPMI8226 and U266 cells treated with cambinol. Apoptosis and the cell cycle were analyzed via flow cytometry. The expression levels of caspase-3, poly(ADP-ribose) polymerase 1 (PARP), p53, acetylated p53 (Ac-p53), Bcl-2, cyclin D1 and p21 were detected in cells treated with cambinol using western blot analysis. The results demonstrated that cambinol inhibited the proliferation of RPMI8226 and U266 cells in a time- and dose-dependent manner. Increased apoptosis and G1 cell cycle arrest, together with enhanced procaspase-3 degradation and PARP cleavage were identified in cambinol-treated cells compared with controls. Western blotting results also revealed the upregulation of p53 acetylation and p21, as well as the downregulation of Bcl-2 and cyclin D1 in cells treated with cambinol. In conclusion, the present results suggest that cambinol inhibits the proliferation and induces apoptosis in RPMI8226 and U266 cells by regulating acetylation of p53 via the targeting of SIRT1.

Background Acute myeloid leukemia (AML) is a malignant hematological neoplasm of myeloid progenitor cells. Mutations of FLT3 in its tyrosine kinase domain (FLT3-TKD) are found in ~ 8% of patients with AML, with D835Y as the most common substitution. This mutation activates survival signals that drives the disease and is resistant to the first generation FLT3 inhibitors. Development of a highly sensitive method to detect FLT3D835Y is important to direct therapeutic options, predict prognosis, and monitor minimal residual disease in patients with AML. Methods and results In the present study, we developed a highly sensitive FLT3D835Y detection method by using the restriction fragment nested allele-specific PCR technique. The method consists of three steps: 1) initial amplification of DNA samples with PCR primers surrounding the FLT3D835Y mutation site, 2) digestion of the PCR products with restriction enzyme EcoRV that only cleaves the wild type allele, and 3) detection of FLT3D835Y by allele-specific PCR with nested primers. We were able to detect FLT3D835Y with a sensitivity of 0.001% by using purified plasmid DNAs and blood cell DNAs containing known proportions of FLT3D835Y. We analyzed blood cell DNA samples from 64 patients with AML and found six FLT3D835Y-positive cases, two of which could not be detected by conventional DNA sequencing methods. Importantly, the method was able to detect FLT3D835Y in a sample collected from a relapsed patient while the patient was in complete remission with negative MRD determined by flow cytometry. Therefore, our RFN-AS-PCR detected MRD after treatment that was missed by flow cytometry and Sanger DNA sequencing, by conventional methods. Conclusions We have developed a simple and highly sensitive method that will allow for detection of FLT3D835Y at a very low level. This method may have major clinical implications for treatment of AML.

Notch signaling is a highly conserved pathway existed in multicellular organisms. It plays roles in normal human body development, human cancer initiation, progression and metastasis. The Notch negative regulatory region (NRR) is critical for Notch signaling, and cleavage at the S2 site in the NRR ultimately leads to the activation of Notch signaling. To study the function of human NRR1, we expressed the recombinant human NRR1 (rhNRR1) domain in Escherichia coli. After purification, rhNRR1 was obtained with approximately 94% purity according to SDS-PAGE analysis. Furthermore, the polyclonal anti-rhNRR1 serum raised by immunizing mouse with the purified rhNRR1 was able to reduce the generation of active form of Notch1 intracellular domain in HeLa cells, which implied the raised antibody could recognize and bind the natural conformation of Notch1 NRR. Preparation of rhNRR1 by this way is convenient, time-consuming, and could be used to the preparation of anti-NRR1 therapeutic antibody.

BACKGROUND Moringa leaf is licensed as a new food ingredient and is rich in nutrients. However, due to its high cellulose content, the nutrients are difficult to digest and absorb, which limits the product development and application of Moringa leaf. RESULTS We chose four bacterial strains—Trichoderma reesei CICC 2626, Bacillus pumilus CICC 10440, Bacillus subtilis subsp. CICC 21934, and Bacillus sp. CICC 21942—to ferment with Moringa leaf powder. The results of solid fermentation and liquid fermentation show that the solid-state fermentation using Bacillus pumilus CICC 10440 in a 1:60 ratio with Moringa leaf powder produced the greatest amount of soluble protein (285 mg/g, 2.42 times higher than the control) in 24 h. Using a cellulase activity assay and western blotting, we found that Bacillus pumilus CICC 10440 uses endoglucanase to dissolve the cellulose to release nutrients from the Moringa oleifera leaf. We expanded the fermentation scale of the Moringa oleifera leaf in a 200 l fermenter and produced a 50-kg batch of Moringa oleifera product. CONCLUSION The results showing improvement in the nutrient release of Moringa leaf via microbial fermentation are a promising start and provide a basis for the further product development of Moringa leaf.

Major histocompatibility complex (MHC) class I-related chain A/B (MICA/B) is a type of stress-induced molecule that plays an important role in tumor surveillance. MICA/B shares a similar structure with MHC class I molecules, but MICA/B contains a closed cleft, not an open one, in its N-terminal alpha1 domain. The alpha 1 domain was believed to have no roles in antigen presentation, because the closed cleft provides limited space for binding with known molecules, and the cleft of MICA/B have been reported no known functions. To study the possible function of the cleft located in human MICA/B's alpha 1 domain, we attempted to express the human MICB-α1 (hMICB-α1) domain allele protein, which is approximately 20.5 kDa, by utilizing an Escherichia coli (E. coli) secretory pathway. Protein expression was accomplished through the phosphate-limited inducible promoter. After purification using ammonium sulfate precipitation, phenyl hydrophobic Sepharose, SP Sepharose and HisTrap affinity Sepharose, recombinant human MICB-α1 (rhMICB-α1) was obtained with 94.3% purity. The binding capacity of rhMICB-α1 with natural killer group 2, member D (NKG2D) was evaluated in vitro. The results demonstrated that rhMICB-α1 can be prepared through the E. coli secretory pathway. Purified rhMICB-α1 protein was able to functionally bind with NKG2D. This method can be further used to obtain functionally active rhMICB-α1 protein, which can served as the basis for further studies of the possible function of the MICB cleft.

In the fields of drug discovery and protein science, small quantities of proteins are always needed to investigate or validate protein–protein (or protein–small molecule) interactions. Traditional transient or stable expression method to obtain recombinant proteins in eukaryotic systems can be laborious and time-consuming, especially when multiple protein variants are required. Here, we present a fast and convenient method for obtaining small quantities of recombinant human epidermal growth factor receptor (rhEGFR) ectodomain protein, which could be efficiently extended to the expression of other eukaryotic proteins. Human EGFR ectodomain gene was inserted into the plasmid pBMN-GFP and recombinant plasmid was transfected into HEK 293 T cells. In the presence of hygromycin, cells with the integrated human EGFR ectodomain gene were selected and proliferated. rhEGFR ectodomain in cell culture supernatant was purified using serial connected diethylaminoethyl Sepharose column and Ni-NTA Sepharose column. Purity of the final purified rhEGFR ectodomain was over 95% according to SDS-PAGE analysis. Moreover, the purified target protein was biological active via measuring the affinity between the rhEGFR ectodomain and rhEGF. Our method could greatly facilitate research in the areas of protein science, protein structural biology, and drug discovery.