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Objective: To establish a detection method based on Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) that can sensitively detect the second messenger cyclic AMP (cAMP) in the cytoplasm. Methods: The eukaryotic expression vectors of CFTR and YFP-H148Q / I152L were constructed respectively. FRT cells co-expressing CFTR and YFP-H148Q / I152L were obtained by liposome transfection. The expression of CFTR and YFP-H148Q / I152L in FRT cells was observed by an inverted fluorescence microscopy, and flow cytometry was used to detect the purity of cells; The cell model was identified by the fluorescence quenching kinetics test. The validation of the cell model which could screen CFTR modulators was verified by the fluorescence quenching kinetics experiments. The radioimmunoassay was used to detect the cAMP concentration in cytoplasm after adding CFTR activator. Results: The results of the inverted fluorescence microscope showed that CFTR was expressed in the cell membrane and YFP-H148Q / I152L was expressed in the cytoplasm of FRT cells. The FRT cell model stably co-expressing ANO1 and YFP-H148Q / I152L was successfully constructed. The model could screen CFTR modulators, and the slope of fluorescence change and the concentration of CFTR modulators were in a dose-dependent manner. The slope of the fluorescence could reflect the cAMP concentration in the cytoplasm. The cell model could sensitively detect the intracellular cAMP concentration. Conclusion: The cell model could efficiently and sensitively detect the second messenger cAMP concentration in the cytoplasm, and it provided a simple and efficient method for the study of other targets associated cAMP signal.

Biofilm is prevalent in various ecological niches, in which microbial cells interconnect with each other through extracellular polymeric substances including polysaccharides, extracellular DNA, and proteins. When living in biofilms, the microbial cells employ small signalling chemicals as their "language" to communicate mutually, and exhibit remarkable differences in physiology compared to those living in planktonic state. It has been proven that the development of biofilm is subject to the regulation of c-di-GMP, an important second messenger found in prokaryotes. Given its important roles of biofilms in microbial infection, industry application, plant-microbe interactions and environmental pollustion, biofilm is one of frontier research areas in microbiology. This special issue of "Biofilm and c-di-GMP" systematically reviews the current progresses in the multiple research frontiers, including biotechnology, infectious diseases, environmental microbiology and plant pathology, with special focus on the methods and techniques in biofilm research. We hope that the issue will boost the interest of students and young scientists in this exciting area of microbiology.

Yersinia pestis, the cause of plague, is transmitted by flea bite. Y. pestis forms a biofilm in the proventriculus of its flea vector to enhance transmission. Biofilm formation in Y. pestis is positively regulated by the intracellular levels of the second messenger cyclic diguanylate (c-di-GMP). The c-di-GMP in Y. pestis is synthesized by two diguanylate cyclases (DGC), HmsT and HmsD, and degraded by phosphodiesterase (PDE), HmsP. Here we summarized the regulators that modulate c-di-GMP metabolism and biofilm formation in Y. pestis and discussed their regulatory mechanism.

cyclic di-GMP (c-di-GMP) is a universal second messenger in bacterial cells. It regulates various biological processes such as biofilm development, pathogenicity, motility, exopolysaccharide (EPS) production and cell cycle. The second messenger exerts its function by binding to effectors, such as riboswitches and proteins. However, due to the diverse conformations of c-di-GMP, its effectors are hardly to be predicted by homology search. Identification of c-di-GMP effectors is the initial step to investigate its regulatory function in bacterial signal transduction, however, it remains to be a technically difficult task. Here we reviewed the mechanism of biofilm development controlled by c-di-GMP through binding to various types of protein effectors, and summarized the screening strategies, including genetics analysis, protein pull-down combined with LC/MS/MS identification, DRaCALA systematic screening and molecular docking-based prediction. We also summarized experimental methods for verifying protein-c-di-GMP interaction, including isothermal titration calorimetry, surface plasmon resonance, microscale thermophoresis etc. In addition, we discussed the advantages and disadvantages of these strategies and methods. The present review aims to facilitate the future investigations that are focused on regulatory role of novel c-di-GMP effectors.

Bacterial biofilm plays an important role in persistent microbial infection. Delineation of the formation and development of bacterial biofilm would provide a promising strategy to treat recalcitrant infection. c-di-AMP (Cyclic diadenosine monophosphate) is a recently identified second messenger of bacteria and involved in plethora of bacterial activities, including cell growth, cell wall homeostasis, biofilm formation and microbial pathogenicity. Here we review the recent literature pertinent to the role and molecular mechanisms of c-di-AMP in regulating biofilm formation of bacteria. The potential application of c-di-AMP and its related proteins in the development of novel antimicrobial therapeutics has also been discussed.

Cyclic diguanosine monophosphate (c-di-GMP) is a ubiquitous nucleotide second messenger present in a wide variety of bacteria. It regulates many important bacterial physiological functions such as biofilm formation, motility, adhesion, virulence and extracellular polysaccharide synthesis. It binds with many different proteins or RNA receptors, one of which is called riboswitch that is usually located at the 5'-untranslational region (5'-UTR) in some mRNA. Riboswitch usually comprises a specific ligand-binding (sensor) domain (named aptamer domain, AD), as well as a variable domain, termed expression platform (EP), to regulate expression of downstream coding sequences. When a specific metabolite concentration exceeds its threshold level, it will bind to its cognate riboswitch receptor to induce a conformational change of 5'-UTR, leading to modulation of downstream gene expression. Two classes of c-di-GMP-binding riboswitches (c-di-GMP-Ⅰ and c-di-GMP-Ⅱ) have been discovered that bind with this second messenger with high affinity to regulate diverse downstream genes, underscoring the importance of this unique RNA receptor in this pathway. Class Ⅰ c-di-GMP riboswitches are present in a wide variety of bacteria, and are most common in the phyla Firmicutes and Proteobacteria, while class Ⅱ c-di-GMP riboswitches typically function as allosteric ribozymes, binding to c-di-GMP to induce folding changes at atypical splicing site junctions to modulate downstream gene expression. This review introduces the discovery, classification, function, and also the affected downstream genes of c-di-GMP riboswitches.

Sphingosine-1-phosphate (S1P), a bioactive sphingolipid produced by the metabolism of sphingomyelin, regulates cell proliferation, migration, survival and cell-cell contacts. The sphingosine-1- phosphate signaling pathway can regulate the trafficking of lymphocyte, angiogenesis, the progress of cancer and many other cellular functions. The formation of S1P is catalyzed by sphingosine kinases (SPHK), and degraded by lyases(S1PL), therefore S1P level is subject to a dynamic balance in the physiological environment. S1P can act as a second messenger or couple with S1P receptors (S1PR) to exert effects. The targets in the S1P signaling pathway have received considerable attention. Here we review the physiological function and drug development of S1P signaling pathway.

Cyclic diadenosine monophosphate (c-di-AMP), a new second messenger found recently in bacteria, regulates various aspects of bacterial physiology, including cell growth, cell wall homeostasis and virulence. In addition to its functions in bacterial physiology, c-di-AMP represents a putative bacterial secondary signaling molecule sensed by eukaryotic host cells and triggers innate immunity. The level of c-di-AMP in bacteria is regulated by the activities of diadenylate cyclase (DAC) and phosphodiesterases (PDE) , the former harbors a DisA_N domain, and the latter a DHH or DHH/DHHA1 domain. This review gives an overview on metabolic pathway, regulatory mechanism, receptor proteins and biological function of c-di-AMP in bacteria, as well as its application and trends of development.

Cyclic diguanylate (c-di-GMP) is a ubiquitous second messenger present in a wide variety of bacteria, which is responsible for cell differentiation, biofilm formation, pathogenic factor generation, and so on. The level of c-di-GMP in bacteria is regulated by two opposing active domains, diguanylate cyclase (DGC) and phosphodiesterase (PDE), which are present in the same bifunctional protein, and in charge of the synthesis and the degradation of c-di-GMP, respectively. The target of c-di-GMP in the bacterial cell consists of PilZ domain and GEMM riboswitch, the only riboswitch that involved in signal transduction. This article gives an overview of c-di-GMP, focusing on its metabolic pathway, regulatory mechanism, biological function of c-di-GMP, and the synthesis of c-di-GMP analogues and their biological activity.

Signal transduction is one of the most important fields of life science. The discovery of cAMP and description of the second messenger hypothesis by Sutherland is the landmark of signal transduction. In this paper, the background and course of cAMP discovery were demonstrated and at the same time the content and significance of the second messenger hypothesis were illustrated, so scientific contribution of the second messenger hypothesis can be approximately understood.

Objective: To investigate the effect of SP on intracellular free calcium concentration (Ca2+)i in cultured osteoblast., Methods: Effect of SP on intracellular free calcium was measured using cell culturing and laser scanning confocal microscopy with fluo-3/AM as a fluorescence probe., Results: After treated with SP(10(-4) mol/L), 70% of the cells responded to it; the intracellular (Ca2+)i increased markedly [fluorescence intensity increased by (90 +/- 18)%]., Conclusions: These results suggested that calcium may have a role as a intracellular secondary messenger in the action of SP in osteoblast.

Aim and Methods: The present research is performed on Xenopus oocytes injected with rat DRG mRNA. Modulatory effects of SKF38393 and Clonidine on GABAS(A) receptors expressed in Xenopus oocytes was investigated by two-electrodes voltage-clamp technique, and it is compared with the neurons fresh isolated., Results: It is found that SKF38393 and Clonidine inhibitable GABA(A) receptor function obviously by a concentration-dependent manner. There is an interaction between SKF38393- and Clonidine-induced currents. The inhibition of SKF38393 and Clonidine on GABA(A) receptors is noncompetitive and voltage-independent., Conclusions: The results suggest that the inhibition by SKF38393 and clonidine of GABA-activated current might be a result of phosphorylation of GABA(A) receptor following action of second messenger and the thereby mediated intracellular transduction.

Objective: The alterations of second messengers induced by acute hypoxia were studied., Methods: The porcine pulmonary and cerebral arterial endothelial cells (PAEC and CAEC) as well as smooth muscle cells (PASMC and CASMC) were cultured, and the hypoxic alteration of [Ca2+]i, inositol phosphates and cyclic nucleotides were measured., Results: During acute hypoxia both [Ca2+]i and cGMP levels increased and IP3, cAMP remained unchanged in PAEC, while in CAEC the intracellular Ca2+ release, the IP3, cAMP and cGMP also increased significantly. A difference in the alteration of cGMP level was observed between PASMC and CASMC., Conclusions: The heterogeneity of endothelial cells might play a role in the different responses of pulmonary and cerebral vessels to hypoxia.

Ceramide may be generated from many metabolism routes. As a novel second messenger, ceramide plays important roles in a variety of physiologic or pathologic events relating to cellular growth, proliferation, differentiation, apoptosis and injuries. It is expected that new insights into the ceramide pathway would promote our understanding of the complex mechanisms for signal transduction.

By using an inducer of differentiation (HMBA) and blockers of signal pathways, the relationship between two second-messenger pathways (DG-PKC, cAMP-PKA) in inducer-mediated MGc80-3 cell differentiation was studied. Cells were treated with HMBA for 24 h, levels of DG decreased by 64.7%, activity of PKC decreased by 28.7%, while levels of cAMP and rate of it's protein binding increased by 62% and 32.6% respectively (after treated for 48 h, the result was more remarkable). PKA-R II distributed in nuclei. H7 (PKC inhibitor) was used to substitute for HMBA to block the DG-PKC pathway. After treatment for 24 h, the levels of DG and activity of PCK both decreased, while the levels of cAMP increased 1.04 times. On the contrary, PKA inhibitor was added while HMBA was used to induce cell differentiation, cAMP-PKA pathway was blocked, levels of cAMP and rate of it's protein binding decreased. But levels of DG and activity of PKC both increased to the levels of control cells. At this time, PKA-R II distributed only in cytoplasm. These results suggest that harmonious relations of the positive and negative regulation of cAMP-PKA and DG-PKC systems in cells during the proliferation and differentiation of cells. It also showed that positive regulation of PI system may play a leading role in MGc80-3 cells. It let to normal regulation of two signal pathway out of control to remain malignant phenotype of these cells.