Label-free cell based impedance measurements of ZnO nanoparticles—human lung cell interaction: a comparison with MTT, NR, Trypan blue and cloning efficiency assays

Background There is a huge body of literature data on ZnOnanoparticles (ZnO NPs) toxicity. However, the reported results are seen to be increasingly discrepant, and deep comprehension of the ZnO NPs behaviour in relation to the different experimental conditions is still lacking. A recent literature overview emphasizes the screening of the ZnO NPs toxicity with more than one assay, checking the experimental reproducibility also versus time, which is a key factor for the robustness of the results. In this paper we compared high-throughput real-time measurements through Electric Cell-substrate Impedance-Sensing (ECIS®) with endpoint measurements of multiple independent assays. Results ECIS-measurements were compared with traditional cytotoxicity tests such as MTT, Neutral red, Trypan blue, and cloning efficiency assays. ECIS could follow the cell behavior continuously and noninvasively for days, so that certain long-term characteristics of cell proliferation under treatment with ZnO NPs were accessible. This was particularly important in the case of pro-mitogenic activity exerted by low-dose ZnO NPs, an effect not revealed by endpoint independent assays. This result opens new worrisome questions about the potential mitogenic activity exerted by ZnO NPs, or more generally by NPs, on transformed cells. Of importance, impedance curve trends (morphology) allowed to discriminate between different cell death mechanisms (apoptosis vs autophagy) in the absence of specific reagents, as confirmed by cell structural and functional studies by high-resolution microscopy. This could be advantageous in terms of costs and time spent. ZnO NPs-exposed A549 cells showed an unusual pattern of actin and tubulin distribution which might trigger mitotic aberrations leading to genomic instability. Conclusions ZnO NPs toxicity can be determined not only by the intrinsic NPs characteristics, but also by the external conditions like the experimental setting, and this could account for discrepant data from different assays. ECIS has the potential to recapitulate the needs required in the evaluation of nanomaterials by contributing to the reliability of cytotoxicity tests. Moreover, it can overcome some false results and discrepancies in the results obtained by endpoint measurements. Finally, we strongly recommend the comparison of cytotoxicity tests (ECIS, MTT, Trypan Blue, Cloning efficiency) with the ultrastructural cell pathology studies. Graphic Abstract

Large-Scale Preparation of Highly Stable Recombinant Human Acidic Fibroblast Growth Factor in Escherichia coli BL21(DE3) plysS Strain

In this study, the optimum human aFGF gene encoding haFGF135 was cloned in pET3c and transferred to Escherichia coli BL21(DE3) plysS. To enhance the yield of fermentation and the expression level of the target protein, the fermentation parameters, including temperature, pH, dissolved oxygen, glucose concentration, ammonium chloride concentration, induction time, and inducer (IPTG) concentration, were optimized. The optimized fermentation parameters were used in large-scale fermentation (30 L). Ion-exchange and heparin-affinity column chromatography techniques were used for separation and purification of rhaFGF135 protein. HPLC, isoelectric focusing electrophoresis, and mass spectrometry were used to detect the purity, isoelectric point, and molecular weight and peptide map of rhaFGF135 protein, respectively. Mitogenic activity of rhaFGF135 protein was detected in NIH-3T3 cells and a full-thickness injury wound diabetic rat model. The production and expression level of rhaFGF135 in the 30-L scale fermentation reached 80.4 ± 2.7 g/L culture and 37.8% ± 1.8%, respectively. The RP-HPLC and SDS-PAGE purity of the final rhaFGF135 product almost reached 100%, and the final pure protein yield was 158.6 ± 6.8 mg/L culture. Finally, the cell and animal experiments showed that rhaFGF135 retained a potent mitogenic activity. The large-scale process of rhaFGF135 production reported herein is relatively stable and time-saving, and thus, it can be used as an efficient and economic strategy for the synthesis of rhaFGF135 at the industrial level.

Isolation, Purification and Characterization of Novel Phytomitogen from Nigella sativa Seeds

The objective of the present study was to extract and purified proliferative factor for peripheral blood lymphocytes from Nigella sativa seeds. Dimeric 44, 32 and 26 -kDa glucosamine-specific lectins were extracted and purified from seeds of N. sativa L. by using different ionic strength solvents (deionized distilled water, phosphate buffer slain and 0.1 M HCl) purification procedure included, precipitation in saturated ammonium sulphate, then Sephadex G-100 gel filtration was used for further purification, finally electrophoresis by polyacrylamide gel to estimate the molecular weight in the presence of sodium dodecylsulphate. Single absorbance peak at 280 nm was recorded in each extracted method. The temperature and pH value on mitogenic activity was studied in temperature ranged from 4°C-60°C, the mitogenic activity reduced with gradual increase of temperatures, 50% of activity lost at temperature greater than 40°C, while the optimum pH for mitogenic activity ranged from 5-9, at pH value less than 3, and greater than 12 the activity disappeared. Interestingly, lectin extracted from N. sativa seeds have high activity especially that extracted with 0.1 M HCl and showed stability at temperature from 4°C to 10°C and pH from 5 to 9. The pure lectin is a homodimeric molecule of 26 kDa with subunit molecular mass of 12.9 kDa. The novel lectin exceed the PHA-Sigma in mitogenic activity.

Insight into the roles of hepatoma derived growth factor related protein-3 under physiological and pathological conditions

Hepatoma derived growth factor related protein-3 (HRP-3) is a HDGF growth factor family member that is expressed mainly in nervous tissues. It shares structural similarities with HDGF but differs in its expression and scope of action. It has recently attracted more attention due to its variable roles. HRP-3 was originally reported as a mitogenic factor. However, over the last decade, additional functions for this growth factor have been uncovered. In addition to its mitogenic activity, other physiological functions were discovered including those related to proliferation, differentiation, and maintenance of neurons, presenting it as a neurotrophic and neuroprotective growth factor. Interestingly, HRP-3 was also shown to be involved in the pathogenesis of certain tumors via its mitogenic, angiogenic, and antiapoptotic effects. Based on this, HRP-3 represents a molecule that can be targeted for the treatment of cancer and various neurodegenerative diseases.