Directly Evolved AlkS-Based Biosensor Platform for Monitoring and High-Throughput Screening of Alkane Production.

Biosynthetic alkane using acyl-ACP aldehyde reductase (AAR) and aldehyde-deformylating oxygenase (ADO) from cyanobacteria is considered a promising alternative for the production of biofuels and chemical feedstocks. However, the lack of suitable screening methods to improve the catalytic efficiency of AAR and ADO has hindered further improvements in alkane production. Herein, a novel alkane biosensor was developed based on transcriptional factor AlkS by directed evolution, which shows sensitive dynamic response curves for exogenous long-chain alkanes as well as in situ monitoring of endogenously produced alkanes. The evolved biosensor enables high-throughput screening of alkane-producing strains from the AAR and ADO mutant library, which led to a 13-fold increase in the production of long-chain alkanes, including a 22-fold increase of C15. This study is the first to improve the alkane production through biosensors, which provides a good reference for the establishment of microbial cell factories for alkane production.

A Review of the Biological Activities of Heterocyclic Compounds Comprising Oxadiazole Moieties.

The oxadiazole core is considered a privileged moiety in many medicinal chemistry applications. The oxadiazole class includes 1,2,3-oxadiazole, 1,2,4-oxadiazole, 1,3,4-oxadiazole, and 1,2,5-oxadiazole. Compounds bearing an oxadiazole ring show a wide range of biological activities, such as anticancer, antibacterial, anti-inflammatory, anti-malarial, and insecticidal properties. Among oxadiazoles, the 1,3,4-oxadiazole has been the most widely explored moiety in medicinal chemistry research. This review is primarily focused on the anticancer, antibacterial, and anti-inflammatory activities of compounds containing 1,2,4-oxadiazole, 1,3,4-oxadiazole and 1,2,5-oxadiazole reported in the last five years.

Monitoring arsenic using genetically encoded biosensors in vitro: The role of evolved regulatory genes.

Toxic pollutant (TP) detection in situ using analytical instruments or whole-cell biosensors is inconvenient. Designing and developing genetically coded biosensors in vitro for real-world TP detection is a promising alternative. However, because the bioactivity and stability of some key biomolecules are weakened in vitro, the response and regulation of reporter protein become difficult. Here, we established a genetically encoded biosensor in vitro with an arsenical resistance operon repressor (ArsR) and GFP reporter gene. Given that the wildtype ArsR did not respond to arsenic and activate GFP expression in vitro, we found, after screening, an evolved ArsR mutant ep3 could respond to arsenic and exhibited an approximately 3.4-fold fluorescence increase. Arsenic induced expression of both wildtype ArsR and ep3 mutant in vitro, however, only ep3 mutant regulated the expression of reporter gene. Furthermore, the effects of cell extracts, temperature, pH, incubation, and equilibrium time were investigated, and the equilibration of reaction mixtures for 30 min at 37 °C was found to be essential for in vitro arsenic detection prior to treatment with arsenic. Based on our data, we established a standard procedure for arsenic detection in vitro. Our results will facilitate the practical application of genetically encoded biosensors in TP monitoring.

Development of dual-fluorescence cell-based biosensors for detecting the influence of environmental factors on nanoparticle toxicity.

With the expanding use of engineered nanoparticles (NPs), development of a high-throughput, sensitive method for evaluating NP safety is important. In this study, we developed cell-based biosensors to efficiently and conveniently monitor NP toxicity. The biosensor cells were obtained by transiently transfecting human cells with biosensor plasmids containing a mCherry gene regulated by an inducible promoter [an activator protein 1 (AP-1) promoter, an interleukin 8 (IL8) promoter, or a B cell translocation gene 2 (BTG2) promoter], with an enhanced green-fluorescent protein gene driven by the cytomegalovirus promoter as the internal control. After optimizing flow cytometric analysis, these dual-fluorescence cell-based biosensors were capable of accurately and rapidly detecting NP toxicity. We found that the responses of AP-1, BTG2, and IL8 biosensors in assessing the toxicity of silver nanoparticles (Ag NPs) showed good dose-related increases after exposure to Ag NPs and were consistent with data acquired by conventional assays, such as western blot, real-time polymerase chain reaction, and immunofluorescence. Further investigation of the effects of environmental factors on Ag NP toxicity revealed that aging in water, co-exposure with fulvic acid, and irradiation with ultraviolet A light could affect Ag NP-induced biosensor responses. These results indicated that these novel dual-fluorescence biosensors can be applied to accurately and sensitively monitor NP toxicity.

Insights into the Ecotoxicity of Silver Nanoparticles Transferred from Escherichia coli to Caenorhabditis elegans.

Previous studies have indicated that engineered nanomaterials can be transferred through the food chain. However, their potential ecotoxicity to the environment is not fully understood. Here, we systematically evaluated the physiological behavior and toxicity of polyvinylpyrrolidone (PVP)-coated silver nanoparticles (AgNPs) using a food chain model from Escherichia coli (E. coli) to Caenorhabditis elegans (C. elegans). Our results demonstrated that AgNPs accumulated in E. coli could be transferred to the C. elegans, and AgNPs were clearly distributed in the gut lumen, subcutaneous tissue and gonad. After being transferred to C. elegans through the food chain, the accumulated AgNPs caused serious toxicity to the higher trophic level (C. elegans), including effects on germ cell death, reproductive integrity and life span. Relative to larger particles (75 nm), small AgNPs (25 nm) more easily accumulated in the food chain and exhibited a stronger toxicity to the higher trophic level. More importantly, both the AgNPs that had accumulated in C. elegans through the food chain and the resulting impairment of germ cells could be transferred to the next generation, indicating that AgNP can cause genetic damage across generations. Our findings highlight that nanomaterials pose potential ecotoxicity to ecosystems via transport through the food chain.

Investigating the environmental factors affecting the toxicity of silver nanoparticles in Escherichia coli with dual fluorescence analysis.

Flow cytometric investigation of the toxic effects of nanoparticles on bacteria is highly challenging and not sensitive due to the interference of aggregated nanoparticles: aggregated nanoparticles and bacteria are similar in size. In this study, an optimized dual fluorescence flow cytometric analysis was developed using PI-Lac::GFP (propidium iodide stained Escherichia coli (lac::GFP)) to monitor the toxicity of silver nanoparticles (AgNPs). As compared with single fluorescence analysis, the dual fluorescence analysis enabled more accurate evaluation of the toxic effects of AgNPs. We used this dual fluorescence analysis to investigate how AgNPs toxicity was affected by two typical environmental factors, divalent metal ions and surfactants. Our data revealed that Cu(2+) and SDS significantly enhanced the toxicity of AgNPs in a dose-dependent manner. SDS enhanced the toxicity of both AgNPs and Ag(+) ions, whereas Cu(2+) increased the toxicity of AgNPs but not dissolved Ag(+) ions. Our results suggest that this dual fluorescence analysis can be used to evaluate the toxicity of AgNPs accurately and sensitively.

Evolved bacterial biosensor for arsenite detection in environmental water.

Arsenic, a ubiquitous presence in the biosphere, often occurs from both natural and anthropogenic sources. Bacterial biosensors based on genetically engineered bacteria have promising applications in detecting the chemical compound and its toxicity. However, most of the bacteria biosensors take advantage of the existing wild-type substrate-induced promoters, which are often low in specificity, affinity and sensitivity, and thus limiting their applications in commercial or field use. In this study, we developed an in vivo evolution procedure with a bidirectional selection scheme for improving the sensitivity of an arsenite-responsive bacterial biosensor through optimization of the inducible operon. As a proof of concept, we evolved the arsenite-induced arsR operon for both low background and high expression through three successive rounds of fluorescence activated cell sorting (FACS) with bidirectional strategy. An arsR operon variant with 12-fold higher activity over the control was isolated, confirming multiple rounds of construction and screening of mutation library, as described here, can be efficiently applied to bacterial biosensor optimization. The evolved arsenite-responsive biosensor demonstrated an excellent performance in the detection of low trace arsenite in environmental water. These results indicate that the technologies of directed evolution could be used to improve the performance of bacterial biosensors, which will be helpful in promoting the practical application of bacterial biosensors.

Synergistic effects induced by a low dose of diesel particulate extract and ultraviolet-A in Caenorhabditis elegans: DNA damage-triggered germ cell apoptosis.

Diesel exhaust has been classified as a potential carcinogen and is associated with various health effects. A previous study showed that the doses for manifesting the mutagenetic effects of diesel exhaust could be reduced when coexposed with ultraviolet-A (UVA) in a cellular system. However, the mechanisms underlying synergistic effects remain to be clarified, especially in an in vivo system. In the present study, using Caenorhabditis elegans (C. elegans) as an in vivo system we studied the synergistic effects of diesel particulate extract (DPE) plus UVA, and the underlying mechanisms were dissected genetically using related mutants. Our results demonstrated that though coexposure of wild type worms at young adult stage to low doses of DPE (20 µg/mL) plus UVA (0.2, 0.5, and 1.0 J/cm2) did not affect worm development (mitotic germ cells and brood size), it resulted in a significant induction of germ cell death. Using the strain of hus-1::gfp, distinct foci of HUS-1::GFP was observed in proliferating germ cells, indicating the DNA damage after worms were treated with DPE plus UVA. Moreover, the induction of germ cell death by DPE plus UVA was alleviated in single-gene loss-of-function mutations of core apoptotic, checkpoint HUS-1, CEP-1/p53, and MAPK dependent signaling pathways. Using a reactive oxygen species (ROS) probe, it was found that the production of ROS in worms coexposed to DPE plus UVA increased in a time-dependent manner. In addition, employing a singlet oxygen (1O2) trapping probe, 2,2,6,6-tetramethyl-4-piperidone, coupled with electron spin resonance analysis, we demonstrated the increased 1O2 production in worms coexposed to DPE plus UVA. These results indicated that UVA could enhance the apoptotic induction of DPE at low doses through a DNA damage-triggered pathway and that the production of ROS, especially (1)O2, played a pivotal role in initiating the synergistic process.