Cyclic dinucleotides are an expanding class of signaling molecules that control many aspects of bacterial physiology. A synthase for cyclic AMP-GMP (cAG, also referenced as 3′-5′, 3′-5′ cGAMP) called DncV is associated with hyperinfectivity of Vibrio cholerae but has not been found in many bacteria, raising questions about the prevalence and function of cAG signaling. We have discovered that the environmental bacterium Geobacter sulfurreducens produces cAG and uses a subset of GEMM-I class riboswitches (GEMM-Ib, Genes for the Environment, Membranes, and Motility) as specific receptors for cAG. GEMM-Ib riboswitches regulate genes associated with extracellular electron transfer; thus cAG signaling may control aspects of bacterial electrophysiology. These findings expand the role of cAG beyond organisms that harbor DncV and beyond pathogenesis to microbial geochemistry, which is important to environmental remediation and microbial fuel cell development. Finally, we have developed an RNA-based fluorescent biosensor for live-cell imaging of cAG. This selective, genetically encodable biosensor will be useful to probe the biochemistry and cell biology of cAG signaling in diverse bacteria.

RNA-based fluorescent (RBF) biosensors have been applied to detect a variety of metabolites in vitro and in live cells. They are designed by combining the ligand sensing domain of natural riboswitches with in vitro selected fluorogenic aptamers. Different biosensor topologies have been developed to accommodate the diversity of riboswitch structures. Here we show that circular permutation of the riboswitch ligand sensing domain also gives functional biosensors, using the SAM-I riboswitch as our model. We reveal that this design can enhance fluorescence turn-on and ligand binding affinity compared to the non-permuted topology.

Functionalized multiwalled carbon nanotubes (f-MWCNTs) can serve as the pseudostationary phase (PSP) for the capillary EKC separation of non-steroidal anti-inflammatory drugs (NSAIDs). To increase their hydrophilicity, we treated MWCNTs, with a sonochemical process in a concentrated nitric/sulfuric acid mixture. The oxidized MWCNTs were then characterized by FT-IR, transmission electron microscopy, and X-ray photoelectron spectroscopy. We evaluated the potential of the PSP and the effects of buffer composition, pH, addition of organic modifier, and injection temperature on the NSAID separation. The PSP created a network structure of pi-pi interactions, hydrophobic forces, hydrogen bonding, and electrostatic interactions to separate NSAIDs, providing a different separation mode from SDS micelles. We achieved complete separation of six NSAIDs using a mixture of a borate buffer (75 mM, pH 10) with methanol (5%, v/v) containing 0.02 mg/mL f-MWCNTs, an applied voltage of +12 kV and detection at 214 nm. Better precision was obtained with a low injection temperature. The method was also satisfactorily applied to the analysis of NSAIDs spiked into a urine sample.

Biodegradable polymers have potential as a scaffold material for making small diameter artery bypass grafts. To resist thrombosis, maintain biocompatibility and enhance the remodeling of the grafts, it is crucial to modify polymer scaffolds so that the grafts have antithrombogenic capacity and allow cell infiltration. In this study, two methods of aminolysis on electrospun poly-L-lactide-co-caprolactone (PLCL) microfiber vascular grafts are compared: plasma treatment method and Fmoc-PEG-diamine insertion method. Both methods successfully inserted amino groups on the polymer graft for heparin conjugation. However, plasma treatment resulted in significantly higher initial heparin density and higher heparin stability on PLCL microfibers than Fmoc-PEG-diamine treatment. In addition, mechanical testing demonstrated that the plasma treatment method maintained PLCL microfiber tensile strength after heparin conjugation. Fmoc-PEG-diamine insertion method compromised the mechanical property due to partial fiber melting and structure disruption. Subcutaneous implantation of the grafts in a rat model showed that heparin coating with both methods promoted cell infiltration. This study provides a rationale to optimize the biomolecule conjugation on electrospun PLCL scaffolds, and will have applications in tissue engineering vascular grafts and other tissues.

A novel pseudostationary phase (PSP) of multiwalled carbon nanotubes (MWCNTs) dispersed with sodium dodecylbenzenesulfonate (NaDDBS) was used for the EKC separation of nucleotides. NaDDBS has a long hydrophobic chain and a benzylsulfonate group. It suspends more MWCNTs (about 100-fold) than SDS, and the π-π interaction between the benzene ring of NaDDBS and MWCNTs prolongs the slurry suspension time. Using NaDDBS as a surfactant can reduce the required amount of MWCNTs and decrease the baseline noise. To produce a stable suspension, the optimum ratio (w/w) of MWCNTs to NaDDBS was investigated with turbidimetry. In this context, several parameters affecting EKC separation were studied, including buffer pH, composition, concentration, and the organic modifier. Use of NaDDBS (8 mg/L)/MWCNTs (0.8 mg/L) as the PSP in a phosphate buffer (30 mM, pH 8) yielded complete resolution of seven geometric isomers of a nucleoside monophosphate. In stacking mode, with 10% MeOH in the sample plug, the mixture of nucleoside mono-, di-, and tri-phosphates was satisfactorily separated in phosphate buffer (50 mM, pH 9). The results indicate that nucleotides with bases containing more electron-withdrawing groups interact more strongly with MWCNTs. The system has been used to separate oligonucleotides, and to analyze nucleotides in a complex matrix sample.