Therapeutic effect and metabolic fingerprinting of triple-negative breast cancer cells following exposure to a novel pH-responsive, gambogic acid-loaded micelle.

Triple-negative breast cancer (TNBC) is a subtype of breast cancer with a poor prognosis and lacks effective therapeutic targets. The use of gambogic acid (GA), a class of active ingredients in traditional Chinese medicine with anti-tumour potential, is limited in tumour therapy owing to its drawbacks and unclear organ toxicity. In this study, we used the pH-responsive amphiphilic block copolymer, PEOz-PCL, to create nanodrugs for GA delivery to MDA-MB-231 cells. The pH-responsive GA-loaded micelles were prepared through nanoprecipitation with a more homogeneous size. The average particle size was 42.29 ± 1.74 nm, and the zeta potential value was 9.88 ± 0.17 mV. The encapsulation rate was 85.06%, and the drug loading rate was 10.63%. The process was reproducible, and sustained release reached 80% in 96 h at acid pH 5.0. Furthermore, cellular tests using CCK-8, TUNEL, and flow cytometry revealed that pH-responsive GA-loaded micelles killed MDA-MB-231 cells more effectively and had much higher activity and targeting compared with free drugs. Metabolomic analysis of the changes in differential metabolites revealed that pH-responsive GA-loaded micelles may inhibit TNBC cells by causing amino acid anabolism, nucleotide metabolism, and glucose metabolism, as well as by affecting their energy sources. The study outcomes will help understand the mechanism of action and the therapeutic efficacy of pH-responsive GA-loaded micellesin vivo.

Engineering thermophilic Geobacillus thermoglucosidasius for riboflavin production.

The potential advantages for fermentation production of chemicals at high temperatures are attractive, such as promoting the rate of biochemical reactions, reducing the risk of contamination and the energy consumption for fermenter cooling. In this work, we de novo engineered the thermophile Geobacillus thermoglucosidasius to produce riboflavin, since this bacterium can ferment diverse carbohydrates at an optimal temperature of 60°C with a high growth rate. We first introduced a heterogeneous riboflavin biosynthetic gene cluster and enabled the strain to produce detectable riboflavin (28.7 mg l-1 ). Then, with the aid of an improved gene replacement method, we preformed metabolic engineering in this strain, including replacement of ribCGtg with a mutant allele to weaken the consumption of riboflavin, manipulation of purine pathway to enhance precursor supply, deletion of ccpNGtg to tune central carbon catabolism towards riboflavin production and elimination of the lactate dehydrogenase gene to block the dominating product lactic acid. Finally, the engineered strain could produce riboflavin with the titre of 1034.5 mg l-1 after 12-h fermentation in a mineral salt medium, indicating G. thermoglucosidasius is a promising host to develop high-temperature cell factory of riboflavin production. This is the first demonstration of riboflavin production in thermophilic bacteria at an elevated temperature.

A novel signal transduction system for development of uric acid biosensors.

Uric acid (UA) is an important biomarker for clinical diagnosis. Here, we present a novel signal transduction system for the development of UA biosensors with the characteristics of stability and ease-of-use. In this system, bacterial allosteric transcription factor HucR was used as the bio-recognition element, and the competition between HucR and the restriction endonuclease HindIII-HF to bind to the designed DNA template was employed to enable signal transduction of UA recognized by HucR. The presence of UA can induce conformational change of HucR, which dissociates HucR from the designed DNA template, allowing the access of the competitor HindIII-HF to cut this DNA template. Thus, the signal of UA recognized by HucR is transduced to easily detectable DNA signal. As proof-of-concept, we demonstrated two UA biosensors by coupling this signal transduction system with real-time quantitative PCR (RT-qPCR) and amplified luminescent proximity homogeneous assay (Alpha), respectively. The RT-qPCR-based UA biosensor has a detection limit of 5 nM with a linear range up to 300 nM UA; Alpha-based UA biosensor has a detection limit of 30 nM with a linear range of 100 nM-10 µM. Moreover, the robustness of both biosensors was verified by reliably detecting UA present in a human serum sample. Altogether, the novel UA biosensors developed in this work hold great potential for clinical application.