Efficient isolation and quantification of circulating tumor cells in non-small cell lung cancer patients using peptide-functionalized magnetic nanoparticles.

BACKGROUND: Circulating tumor cells (CTCs) carry a wealth of information on primary and metastatic tumors critical for enhancing the understanding of the occurrence, progression and metastasis of non-small cell lung cancer (NSCLC). However, the low sensitivity of traditional tumor detection methods limits the application of CTCs in the treatment and disease surveillance of NSCLC. Therefore, CTCs isolation and detection with high sensitivity is highly desired especially for NSCLC patients, which is significant because of high occurrence and mortality. While it is very challenging because of the lower expression of CTC positive biomarkers such as epithelial cell adhesion molecules and cytokeratins (EpCAM and CKs), herein we report a method based on peptide-functionalized magnetic nanoparticles with high CTC capture efficiency, which demonstrates superiority in NSCLC clinical applications. METHODS: For analysis and comparison of the peptide-functionalized magnetic nanoparticles (TumorFisher, Nanopep Corp.) and the antibody-modified magnetic beads (CellSearch, Janssen Diagnostics, LLC), two NSCLC cell lines, A549 and NCI-H1975 were chosen to measure the binding affinity and capture efficiency. In order to compare the effect of the clinical application of these two detection systems, 7 early stage patients with NSCLC were enrolled in this study. To further explore the clinical utility of CTC counting in different stages, 81 NSCLC patients in stage I-IV were enrolled for CTC enumeration and statistical analysis. RESULTS: The binding affinities of the recognition peptide to A549 and NCI-H1975 are 76.7%±11.0% and 70.1%±4.8%, respectively, which is similar with the positive control group (anti-EpCAM antibodies). CTCs were captured in 5/7 (71.4%) of early stage NSCLC patients with NSCLC in TumorFisher system, which is higher than CellSearch, and the false negative of TumorFisher is much lower than CellSearch. In a larger clinical cohort, the CTC numbers of NSCLC patients varied in different stages and the overall detection rate of TumorFisher was 59/81 (72.8%), with the similar proportion in stage I (21/29, 72.4%), II (17/22, 77.3%) and III (16/21, 76.2%). CONCLUSIONS: Highly efficient CTC isolation technique based on peptide-magnetic nanoparticles was firstly applied in NSCLC patients. Compared with the antibody-based the technique, the higher CTC detection rates (71.4%) in NSCLC patient blood samples were demonstrated for the patients in different stages, I-IV, especially in early stages. This indicates the feasibility of the clinical utility of this new technique in early stage screening, prognosis and therapy evaluation of NSCLC.

Isolating promoters from Corynebacterium ammoniagenes ATCC 6871 and application in CoA synthesis.

BACKGROUND: Corynebacterium ammoniagenes is an important industrial organism that is widely used to produce nucleotides and the potential for industrial production of coenzyme A by C. ammoniagenes ATCC 6871 has been shown. However, the yield of coenzyme A needs to be improved, and the available constitutive promoters are rather limited in this strain. RESULTS: In this study, 20 putative DNA promoters derived from genes with high transcription levels and 6 promoters from molecular chaperone genes were identified. To evaluate the activity of each promoter, red fluorescence protein (RFP) was used as a reporter. We successfully isolated a range of promoters with different activity levels, and among these a fragment derived from the upstream sequence of the 50S ribosomal protein L21 (Prpl21) exhibited the strongest activity among the 26 identified promoters. Furthermore, type III pantothenate kinase from Pseudomonas putida (PpcoaA) was overexpressed in C. ammoniagenes under the control of Prpl21, CoA yield increased approximately 4.4 times. CONCLUSIONS: This study provides a paradigm for rational isolation of promoters with different activities and their application in metabolic engineering. These promoters will enrich the available promoter toolkit for C. ammoniagenes and should be valuable in current platforms for metabolic engineering and synthetic biology for the optimization of pathways to extend the product spectrum or improve the productivity in C. ammoniagenes ATCC 6871 for industrial applications.

A metallo-keratinase from a newly isolated Acinetobacter sp. R-1 with low collagenase activity and its biotechnological application potential in leather industry.

Microbial keratinase is a well-recognized enzyme that can specifically degrade insoluble keratins. A keratinase-producing bacterium was isolated from a duck ranch soil and identified as Acinetobacter sp. R-1 based on the biochemical characteristics and 16S rDNA gene sequencing. It showed high keratinase activity and low collagenase activity. The keratinase was purified to electrophoretic homogeneity with 6.69% recovery, 2.68-fold purification and an estimated molecular weight of 25 kDa. Additionally, the keratinase showed optimal activity at 50 °C and pH11. Keratinase activity of Acinetobacter sp. significantly increased in the presence of Li(+), Na(+), and Ca(2+), while it was completely inhibited by EDTA, indicating it was a metallo-keratinase. Moreover, the crude keratinase from Acinetobacter sp. R-1 could thoroughly depilate goat skin and simultaneously modify the wool surface, which indicated its applicable potential in leather and textile industries.