Detection of molecular oxygen using nanosecond-laser-induced plasma.

Molecular oxygen (O2) concentration is measured by employing nanosecond laser-induced plasmas (ns-LIP) over a broad temperature spectrum ranging from 300 K to 1000 K, in the presence of an additional oxygen-containing molecule, CO2. Typically, emission spectra emanating from ns-LIP are devoid of molecular information, as the ns-LIP causes the dissociation of molecular species within the plasma. However, atomic oxygen absorption lines that momentarily appear at 777 nm in the broadband emission from the early-stage plasma are determined to be highly sensitive to the O2 mole fraction but negligibly affected by the CO2 mole fraction. The atomic O absorbing the plasma emission originates from the O2 adjacent to the plasma: robust UV radiation from the early-stage plasma selectively dissociates adjacent O2, exhibiting a relatively low photodissociation threshold, thus generating the specific meta-stable oxygen capable of absorbing photons at 777 nm. A theoretical model is introduced, explicating the formation of the meta-stable O atom from adjacent O2. To sustain the UV radiation from the plasma under high-temperature and low-density ambient conditions, a preceding breakdown is triggered by a split laser pulse (532 nm). This breakdown acts as a precursor, seeding electrons to intensify the inverse-Bremsstrahlung photon absorption of the subsequent laser pulse (1064 nm). Techniques such as proper orthogonal decomposition (POD) and support vector regression (SVR) are employed to precisely evaluate the O2 mole fraction (<1% uncertainty), by analyzing the short-lived (<10 ns) O2-indicator depicted in the early-stage plasma.

Expression of Antimicrobial Peptide (AMP), Cecropin B, in a Fused Form to SUMO Tag With or Without Three-Glycine Linker in Escherichia coli and Evaluation of Bacteriolytic Activity of the Purified AMP.

Current antibiotics have limited action mode, which makes it difficult for the antibiotics dealing with the emergence of bacteria resisting the existing antibiotics. As a need for new bacteriolytic agents alternative to the antibiotics, AMPs have long been considered substitutes for the antibiotics. Cecropin B was expressed in a fusion form to six-histidine and SUMO tags in Escherichia coli. Six-histidine tag attached to SUMO was for purification of SUMO-cecropin B fusion proteins and removal of the SUMO tag from cecropin B. Chimeric gene was constructed into pKSEC1 vector that was designed to be functional in both Escherichia coli and chloroplast. To maximize translation of the fusion protein, sequences were codon-optimized. Four different constructs were tested for the level of expression and solubility, and the construct with a linker, 6xHisSUMO3xGly-cecropin B, showed the highest expression. In addition, cleavage of the SUMO tag by SUMOase in the three fusion constructs which have no linker sequence (3xGly, three glycines) was not as efficient as the construct with the linker between SUMO and cecropin B. The cleaved cecropin B showed bacteriolytic activity against Bacillus subtilis at a concentration of 0.0625 µg/µL, while cecropin B fused to SUMO had no activity at a higher concentration, 0.125 µg/µL. As an expression system for AMPs in prokaryotic hosts, the use of tag proteins and appropriate codon-optimization strategy can be employed and further genetic modification of the fusion construct should help the complete removal of the tag proteins from the AMP in the final step of purification.

Synthesis and Biological Evaluation of BODIPY-PF-543.

Sphingosine-1-phosphate (S1P) regulates the proliferation of various cells and promotes the growth of cancer cells. Sphingosine kinase (SK), which transforms sphingosine into S1P, has two isotypes: SK1 and SK2. To date, both isotypes are known to be involved in the proliferation of cancer cells. PF-543, an SK1 inhibitor developed by Pfizer, strongly inhibits SK1. However, despite its strong SK1 inhibitory effect, PF-543 shows low anticancer activity in vitro. Therefore, additional biological evidence on the anticancer activity of SK1 inhibitor is required. The present study aimed to investigate the intracellular localization of PF-543 and identify its association with anticancer activity by introducing a fluoroprobe into PF-543. Boron-dipyrromethene (BODIPY)-introduced PF-543 has a similar SK1 inhibitory effect as PF-543. These results indicate that the introduction of BODIPY does not significantly affect the inhibitory effect of SK1. In confocal microscopy after BODIPY-PF-543 treatment, the compound was mainly located in the cytosol of the cells. This study demonstrated the possibility of introducing fluorescent material into an SK inhibitor and designing a synthesized compound that is permeable to cells while maintaining the SK inhibitory effect.

Synthesis and Biological Evaluation of PF-543 Derivative Containing Aliphatic Side Chain.

The PF-543 is known as a potent and selective inhibitor of sphingosine kinase (SK) 1 amongst all the SK inhibitors known to date. In a recently reported study by Pfizer on the synthesis of PF-543 derivatives and the SK inhibitory effects, the introduction of propyl moiety into sulfonyl group of PF-543 in the case of 26b revealed an excellent result of 1.7 nM of IC50 of SK1, suggesting the potential substitution of chain structure for benzenesulfonyl structure. In the present work, we aimed for identification of antitumor activity and inhibitory effects of PF-543 derivative containing aliphatic long chain (similar to known SK inhibitors) on SK1. The synthesized compound 2 exhibited an inhibitory effect on SK1 in a manner similar to that of PF-543; the PF-543 derivative manifested similar antitumor activity on HT29, HCT116 (colorectal cancer cell line), and AGS (gastric cancer cell line) cells. Also, from the docking study conducted with PF-543 and compound 2, it was apparent that the aliphatic chain in compound 2 could probably replace benzenesulfonyl structure of PF-543.

A new prokaryotic expression vector for the expression of antimicrobial peptide abaecin using SUMO fusion tag.

BACKGROUND: Despite the growing demand for antimicrobial peptides (AMPs) for clinical use as an alternative approach against antibiotic-resistant bacteria, the manufacture of AMPs relies on expensive, small-scale chemical methods. The small ubiquitin-related modifier (SUMO) tag is industrially practical for increasing the yield of recombinant proteins by increasing solubility and preventing degradation in expression systems. RESULTS: A new vector system, pKSEC1, was designed to produce AMPs, which can work in prokaryotic systems such as Escherichia coli and plant chloroplasts. 6xHis was tagged to SUMO for purification of SUMO-fused AMPs. Abaecin, a 34-aa-long antimicrobial peptide from honeybees, was expressed in a fusion form to 6xHis-SUMO in a new vector system to evaluate the prokaryotic expression platform of the antimicrobial peptides. The fusion sequences were codon-optimized in three different combinations and expressed in E. coli. The combination of the native SUMO sequence with codon-optimized abaecin showed the highest expression level among the three combinations, and most of the expressed fusion proteins were detected in soluble fractions. Cleavage of the SUMO tag by sumoase produced a 29-aa-long abaecin derivative with a C-terminal deletion. However, this abaecin derivative still retained the binding sequence for its target protein, DnaK. Antibacterial activity of the 29-aa long abaecin was tested against Bacillus subtilis alone or in combination with cecropin B. The combined treatment of the abaecin derivative and cecropin B showed bacteriolytic activity 2 to 3 times greater than that of abaecin alone. CONCLUSIONS: Using a SUMO-tag with an appropriate codon-optimization strategy could be an approach for the production of antimicrobial peptides in E.coli without affecting the viability of the host cell.

Synthesis and Biological Evaluation of FTY720 (Fingolimod) Derivatives with Aromatic Head Group as Anticancer Agents.

FTY720 is employed for the treatment of multiple sclerosis and exerts apoptotic effects on various cancers through protein phosphatase 2A (PP2A) activation. In compound 4, the dihydroxy head group of FTY720 was modified into dihydroxy phenyl group. The cell survival in compound 4 treated colorectal and gastric cancer cells was significantly reduced as compared with control, 34.6 and 25.1%, respectively. The docking study of compound 4 showed that the aromatic head group effectively binds to PP2A.

Synthesis of Novel FTY720 Analogs with Anticancer Activity through PP2A Activation.

FTY720 inhibits various cancers through PP2A activation. The structure of FTY720 is also used as a basic structure for the design of sphingosine kinase (SK) inhibitors. We have synthesized derivatives using an amide chain in FTY720 with a phenyl backbone, and then compounds were screened by an MTT cell viability assay. The PP2A activity of compound 7 was examined. The phosphorylation levels of AKT and ERK, downstream targets of PP2A, in the presence of compound 7, were determined. Compound 7 may exhibit anticancer effects through PP2A activation rather than the mechanism by inhibition of SK1 in cancer cells. In the docking study of compound 7 and PP2A, the amide chain of compound 7 showed an interaction with Asn61 that was different from FTY720, which is expected to affect the activity of the compound.