Isolation, Mutagenesis, and Organic Acid Secretion of a Highly Efficient Phosphate-Solubilizing Fungus.

The highly effective phosphate-solubilizing microorganisms are significant for making full use of the potential phosphorus resources in the soil and alleviating the shortage of phosphorus resources. In this study, a phosphate-solubilizing fungus was isolated from wheat and cotton rhizosphere soils in the lower reaches of the Yellow River in China and was identified as Penicillium oxalicum by morphological and ITS sequencing analysis. In order to obtain a fungus with more efficient phosphorus solubilization ability, we tested three positive mutant strains (P1, P2, and P3) and three negative mutant strains (N1, N2, and N3) through low-energy nitrogen ion implantation mutagenesis. Compared with the parental strain, the phosphate-solubilizing capacity of P1, P2, and P3 was enhanced by 56.88%, 42.26%, and 32.15%, respectively, and that of N1, N2, and N3 was weakened by 47.53%, 35.27%, and 30.86%, respectively. Compared with the parental strain, the total amount of organic acids secreted significantly increased in the three positive mutant strains and decreased in the negative mutant strains; the pH of culture medium was significantly lower in the positive mutant strains and higher in the negative mutant strains. The capacity of phosphate-solubilizing fungus to secrete organic acids and reduce the growth-medium pH was closely related to its phosphate-solubilizing ability. The changes in the amount of organic acids secreted by mutants can alter their acidification and phosphate-solubilizing capacity. In conclusion, this study offers a theoretical basis and strain materials for the exploration and application of phosphate-solubilizing fungi.

Novel Peptide-Based Magnetic Nanoparticle for Mesenchymal Circulating Tumor Cells Detection.

The monitoring of circulating tumor cells (CTCs) has recently served as a promising approach for assessing prognosis and evaluating cancer treatment. We have already developed a CTCs enrichment platform by EpCAM recognition peptide-functionalized magnetic nanoparticles (EP@MNPs). However, considering heterogeneous CTCs generated through epithelial-mesenchymal transition (EMT), mesenchymal CTCs would be missed with this method. Notably, N-cadherin, overexpressed on mesenchymal CTCs, can facilitate the migration of cancer cells. Hence, we screened a novel peptide targeting N-cadherin, NP, and developed a new CTCs isolation approach via NP@MNPs to complement EpCAM methods' deficiencies. NP@MNPs had a high capture efficiency (about 85%) of mesenchymal CTCs from spiked human blood. Subsequently, CTCs were captured and sequenced at the single-cell level via NP@MNPs and EP@MNPs, RNA profiles of which showed that epithelial and mesenchymal subgroups could be distinguished. Here, a novel CTCs isolation platform laid the foundation for mesenchymal CTCs isolation and subsequent molecular analysis.

A Microfluidic Chip for Efficient Circulating Tumor Cells Enrichment, Screening, and Single-Cell RNA Sequencing.

Single-cell RNA sequencing on circulating tumor cells (CTCs) proves useful to study mechanisms of tumor heterogeneity, metastasis, and drug resistance. Currently, single-cell RNA sequencing of CTCs usually takes three prerequisite steps: enrichment of CTCs from whole blood, characterization of captured cells by immunostaining and microscopic imaging, and single-cell isolation through micromanipulation. However, multiple pipetting and transferring steps can easily cause the loss of rare CTCs. To address this issue, a novel integrated microfluidic chip for sequential enrichment, isolation, and characterization of CTCs at single-cell level, is developed. And, single CTC lysis is achieved on the same chip. The microfluidic chip includes functions of blood clot filtration, single-cell isolation, identification, and target single-cell lysate collection. By spiking tumor cells into whole blood, it is validated that this microfluidic chip can effectively conduct single-cell CTCs RNA sequencing. The approach lays a solid foundation for the analysis of RNA expression profiling of single-cell CTCs.

Device for whole genome sequencing single circulating tumor cells from whole blood.

Whole-genome sequencing on circulating tumor cells (CTCs) at the single cell level has recently been found helpful for precision medicine, as the oncogenic profiles of single CTCs are useful for discovering oncogenic mutation heterogeneities and guiding/adjusting cancer treatment. To overcome the limits of existing methods of single CTC sequencing, in which CTC enrichment, identification and gene amplification are performed by discrete modules, this study presents a novel method in which all processing steps from blood sample collection to preparation of gene amplification products for sequencers are finished in a single microfluidic chip. This microfluidic chip comprehensively performs blood filtering, CTC enrichment, CTC identification/isolation, CTC lysis and whole genome amplification (WGA) at the single cell level. By sequencing single CTCs from clinical blood samples with pointing key driver and drug-resistance mutations, the novel microfluidic chip was validated to be capable of genetically profiling single CTCs with minimum cell loss/human labor, and more importantly, high accuracy and repeatability, which are crucial factors for promoting clinical application of single CTC sequencing.

A Novel Peptide Probe for Identification of PLS3-Expressed Cancer Cells.

The T-plastin (PLS3) has a significant implication in epithelial-mesenchymal transition (EMT) and breast cancer prognosis. Using one-bead-one-compound library strategy, a novel peptide TP1 (KVKSDRVC) toward PLS3 was screened and exhibited the specificity for identifying PLS3-expressed cancer cells. Moreover, we found Fluorescein isothiocyanate-labeled TP1 (FITC-TP1) could act as a novel probe for EMT-induced cancer cells, preferentially in the leading edge. It also has satisfactory specificity for PLS3-expressed cancer cells spiked in the blood. FITC-TP1 was expected to become a diagnostic tool to identify PLS3-expressed circulating tumor cells and predict prognosis for patients with breast cancer in the future.

Plastin 3 down-regulation augments the sensitivity of MDA-MB-231 cells to paclitaxel via the p38 MAPK signalling pathway.

Plastin 3 (PLS3) overexpression may serve as a marker for predicting chemotherapeutic outcomes in drug-resistant cancer cells, but the mechanism is unclear. Herein, we show that the down-regulation of PLS3 by PLS3 gene silencing augments the sensitivity of MDA-MB-231 triple-negative breast cancer cells to paclitaxel. Interestingly, a low concentration of paclitaxel was able to induce strong apoptosis in the PLS3-silenced cells. Further study revealed that p38 MAPK signalling was responsible for the increased sensitivity to paclitaxel in these cells, as the p38 MAPK inhibitor SB203580 impaired the changes mediated by PLS3 down-regulation in response to paclitaxel. Therefore, our study identifies PLS3 as a potential target for enhancing the p38 MAPK-mediated apoptosis induced by paclitaxel. Unlike paclitaxel, Abraxane was unable to induce strong apoptosis in the PLS3-silenced cells. As PLS3 was found to be involved in the process of endocytosis in breast cancer cells, the reliance of cellular Abraxane uptake on this process may render it not as efficient as paclitaxel in PLS3-depleted tumour cells. The finding that PLS3 could be a critical regulator of paclitaxel sensitivity may have important implications for breast cancer chemotherapy.