Targets for the reduction of antibiotic use in humans in the Transatlantic Taskforce on Antimicrobial Resistance (TATFAR) partner countries.

Unnecessary and inappropriate use of antibiotics in human healthcare is a major driver for the development and spread of antimicrobial resistance; many countries are implementing measures to limit the overuse and misuse of antibiotics e.g. through the establishment of antimicrobial use reduction targets. We performed a review of antimicrobial use reduction goals in human medicine in Transatlantic Taskforce on Antimicrobial Resistance partner countries. On 31 March 2017, the European Centre for Disease Prevention and Control sent a questionnaire to National Focal Points for Antimicrobial Consumption and the National Focal Points for Antimicrobial Resistance in 28 European Union countries, Iceland and Norway. The same questionnaire was sent to the TATFAR implementers in Canada and the United States. Thirty of 32 countries replied. Only nine countries indicated that they have established targets to reduce antimicrobial use in humans. Twenty-one countries replied that no target had been established. However, 17 of these 21 countries indicated that work to establish such targets is currently underway, often in the context of developing a national action plan against antimicrobial resistance. The reported targets varied greatly between countries and can be a useful resource for countries willing to engage in the reduction of antibiotic use in humans.

Binding of GEF-H1 to the tight junction-associated adaptor cingulin results in inhibition of Rho signaling and G1/S phase transition.

The activity of Rho GTPases is carefully timed to control epithelial proliferation and differentiation. RhoA is downregulated when epithelial cells reach confluence, resulting in inhibition of signaling pathways that stimulate proliferation. Here we show that GEF-H1/Lfc, a guanine nucleotide exchange factor for RhoA, directly interacts with cingulin, a junctional adaptor. Cingulin binding inhibits RhoA activation and signaling, suggesting that the increase in cingulin expression in confluent cells causes downregulation of RhoA by inhibiting GEF-H1/Lfc. In agreement, RNA interference of GEF-H1 or transfection of GEF-H1 binding cingulin mutants inhibit G1/S phase transition of MDCK cells, and depletion of cingulin by regulated RNA interference results in irregular monolayers and RhoA activation. These results indicate that forming epithelial tight junctions contribute to the downregulation of RhoA in epithelia by inactivating GEF-H1 in a cingulin-dependent manner, providing a molecular mechanism whereby tight junction formation is linked to inhibition of RhoA signaling.

Histone deacetylase inhibitors up-regulate the expression of tight junction proteins.

Histone deacetylase (HDAC) inhibitors promote cell maturation, differentiation, and apoptosis through changes in gene expression. Differentiated epithelial cells are characterized by apical tight junctions (TJ), which play a role in cell-cell adhesion, polarity, and the permeability barrier function of epithelia. The relationship between cellular differentiation and expression of TJ-associated proteins is not known. Here, we investigated whether HDAC inhibitors affect the expression of TJ proteins in cultured cells by immunoblotting, immunofluorescence, and quantitative real-time, reverse transcription-PCR. We find that the HDAC inhibitor sodium butyrate significantly up-regulates the protein levels of cingulin, ZO-1, and ZO-2 in Rat-1 fibroblasts, cingulin in COS-7 cells, and cingulin and occludin in HeLa cells. Levels of mRNA for cingulin, ZO-1, and ZO-2 are also increased in sodium butyrate-treated Rat-1 fibroblasts. Up-regulation of cingulin is reversible and dose dependent and requires de novo protein synthesis and protein kinase activity, because it is inhibited by cycloheximide and by the protein kinase inhibitor H-7. Up-regulation of TJ proteins by sodium butyrate is linked to the ability of sodium butyrate to inhibit HDAC activity, because suberoylanilide hydroxamic acid, a HDAC inhibitor of a different structural class, also up-regulates cingulin, ZO-1, and ZO-2 expression in Rat-1 fibroblasts. These results indicate that cellular differentiation correlates with kinase-dependent up-regulation of the expression of specific TJ proteins.

Molecular complexity of vertebrate tight junctions (Review).

Separation of distinct body, organ and tissue compartments, and maintenance of epithelial cell polarity require tight junctions (TJ)-cell-cell junctions located in the apicolateral regions of epithelial and endothelial cells. Studies on the protein components of vertebrate TJ have revealed an intricate network of membrane, sub-membrane, cytoskeletal, and signalling molecules. How these molecules functionally interact to provide TJ with their functions, and what roles these molecules play in control of cell growth and differentiation is a fundamental problem in cell biology.

Evidence for a functional interaction between cingulin and ZO-1 in cultured cells.

Cingulin, a protein component of the submembrane plaque of tight junctions (TJ), contains globular and coiled-coil domains and interacts in vitro with several TJ and cytoskeletal proteins, including the PDZ protein ZO-1. Overexpression of Xenopus cingulin in transfected Xenopus A6 cells resulted in the disruption of endogenous ZO-1 localization, suggesting that cingulin functionally interacts with ZO-1. Glutathione S-transferase pull-down experiments showed that a conserved ZO-1 interaction motif (ZIM) at the NH(2) terminus of cingulin is required for cingulin-ZO-1 interaction in vitro. An NH(2)-terminal region of cingulin, containing the ZIM, was sufficient, when fused to coiled-coil sequences, to target transfected cingulin to junctions. However, deletion of the ZIM did not abolish junctional localization of transfected cingulin in A6 cells, suggesting that cingulin can be recruited to TJ through multiple protein interactions. Interestingly, the ZIM was required for cingulin recruitment into ZO-1-containing adherens junctions of Rat-1 fibroblasts, indicating that cingulin junctional recruitment does not require the molecular context of TJ. Cingulin coiled-coil sequences enhanced the junctional accumulation of expressed cingulin head region in A6 cells, but purified recombinant cingulin did not form filaments under physiological conditions in vitro, suggesting that the cingulin coiled-coil domain acts primarily by promoting dimerization.