ABSTRACT
In the present work, we have investigated the effect of pH and mercury ions on the host-guest complex formed between cucurbit[7]uril (Q[7]) and Hoechst 33342 (H33342). 1H NMR, UV-vis and fluorescence spectroscopy revealed that acid/base stimulation could change the binding stoichiometry between Q[7] and H33342. The results suggest that two complexation equilibria (1:1 and 2:1) may exist between H33342 and Q[7] at pH 2.0 and 4.5, respectively. However, a 1:1 host-guest complex was formed between H33342 and Q[7] at pH 7.0 and 10.0. Q[7] shows differential affinities for the protonated and neutral forms of H33342 dye. Moreover, the switching between H33342â2H+@2Q[7](1:2) at pH 4.5 and H33342âH+@Q[7](1:1) at pH 7.0 was reversible. Furthermore, as a metal stimulus, Hg2+ ions could push (i) Q[7] from the piperazine ring to the benzimidazole position with a 1:1 guest-host ratio and (ii) a second Q[7] onto the ethyl position with a 1:2 guest-host stoichiometry. This stimulus response system will have potential applications in the field of molecular switch design.
ABSTRACT
Pseudomonas putida (P. putida) KT2440 is a paradigmatic environmental-bacterium that possesses significant potential in synthetic biology, metabolic engineering and biodegradation applications. However, most genome editing methods of P. putida KT2440 depend on heterologous repair proteins and the provision of donor DNA templates, which is laborious and inefficient. In this report, an efficient cytosine base editing system was established by using cytidine deaminase (APOBEC1), enhanced specificity Cas9 nickase (eSpCas9ppD10A) and the uracil DNA glycosylase inhibitor (UGI). This constructed base editor converts C-G into T-A in the absence of DNA strands breaks and donor DNA templates. By introducing a premature stop codon in target spacers, we successfully applied this system for gene inactivation with an efficiency of 25-100% in various Pseudomonas species, including P. putida KT2440, P. aeruginosa PAO1, P. fluorescens Pf-5 and P. entomophila L48. We engineered an eSpCas9ppD10A-NG variant with a NG protospacer adjacent motif to expand base editing candidate sites. By modifying the APOBEC1 domain, we successfully narrowed the editable window to increase gene inactivation efficiency in cytidine-rich spacers. Additionally, multiplex base editing in double and triple loci was achieved with mutation efficiencies of 90-100% and 25-35%, respectively. Taken together, the establishment of a fast, convenient and universal base editing system will accelerate the pace of future research undertaken with P. putida KT2440 and other Pseudomonas species.
ABSTRACT
OBJECTIVE: To investigate the mechanism of immunologic escape in the tumor microenvironment, study the expression of programmed death 1 ligand-1 (PD-L1) in Tca8113 with treatment of inflammatory factors. METHODS: The expression of PD-L1 treated with inflammatory factors (IL-1beta, IL-2, IL-6, TNF-alpha, IFN-gamma) was detected by flow cytometry (FCM). RESULTS: The expression of PD-L1 in Tca8113 was up-regulated conspicuously with treatment of inflammatory factors (P < 0.05), including: IL-1beta, IL-6, TNF-alpha, IFN-y. And the factors played the role in synergistic effects, most significantly in the groups of IL-1beta + IFN-gamma, TNF-alpha + IFN-gamma and IL-1beta + IL-6 + IFN-gamma + TNF-alpha. But the influence of IL-2 on the expression of PD-L1 was not significant (P > 0.05). CONCLUSION: With the up-regulated expression of PD-L1, the tumor would be escaped more easily from the immunoreactions, while there were an abundance of inflammatory factors in the tumor microenvironment.
