Recent advances in fluorescent protein technology provide a wide variety of biological imaging applications; however current tools for bio-imaging in the Gram-positive bacterium Staphylococcus aureus has necessitated further developments for fluorescence intensity and for a multicolor palette of fluorescent proteins. To enhance the expression of multicolor fluorescent proteins in clinical S. aureus strains, we developed new fluorescent protein expression vectors, containing the blaZ/sodp promoter consisting of the β-lactamase gene (blaZ) promoter and the ribosome binding site (RBS) of superoxide dismutase gene (sod). We found S. aureus-adapted GFP (GFPsa) driven by the blaZ/sodp promoter was highly expressed in the S. aureus laboratory strain RN4220, but not in the clinical strains, MW2 and N315, harboring the endogenous blaI gene, a repressor of the blaZ gene promoter. We therefore constructed a constitutively induced blaZ/sodp promoter (blaZ/sodp(Con)) by introducing substitution mutations into the BlaI binding motif, and this modification allowed enhanced expression of the multicolor GFP variants (GFPsa, EGFP, mEmerald, Citrine, Cerulean, and BFP) as well as codon-optimized reef coral fluorescent proteins (mCherry and AmCyan) in the S. aureus clinical strains. These new fluorescent probes provide new tools to enhance expression of multicolor fluorescent proteins and facilitate clear visualization of clinical S. aureus strains.

To identify individual chromosomes of a frog karyotype by their fluorescence banding patterns, chromosomes were stained with actinomycin D and 4,6-diamidino-2-phenylindole (DAPI) after incorporation of BrdU during the late S-phase. The chromosomes of three Rana species which were selected for this study (R. ridibunda, R. lessonae and R. japonica) showed well-defined late replication bands. The fluorescence patterns obtained were the reverse of those produced by a 4Na-EDTA Giemsa-staining technique. Fluorescence patterns of the two water frog species (R. ridibunda and R. lessonae) were similar to each other, except for the different fluorescence of the centromeric heterochromatin, which gave extremely bright signals in R. ridibunda but no signal in R. lessonae. Experiments also showed differences between the fluorescence patterns of R. lessonae chromosome 13 in the Italian and Luxembourgian populations. These results show that the fluorescence replication banding using actinomycin D and DAPI is very effective in identifying individual frog chromosomes and detecting their structural changes.

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We previously showed that 5-ethynyl-(1-β-D-ribofuranosyl)imidazole-4-carboxamide (1; EICAR) is a potent anti-dengue virus (DENV) compound but is cytotoxic to some cell lines, while its 4-thio derivative, 5-ethynyl-(4-thio-1-β-D-ribofuranosyl)imidazole-4-carboxamide (2; 4′-thioEICAR), has less cytotoxicity but also less anti-DENV activity. Based on the hypothesis that the lower anti-DENV activity of 2 is due to reduced susceptibility to phosphorylation by cellular kinase(s), we investigated whether a monophosphate prodrug of 2 can improve its activity. Here, we first prepared two types of prodrug of 1, which revealed that the S-acyl-2-thioethyl (SATE) prodrug had stronger anti-DENV activity than the aryloxyphosphoramidate (socalled ProTide) prodrug. Based on these findings, we next prepared the SATE prodrug of 4′-thioEICAR 18. As expected, the resulting 18 showed potent anti-DENV activity, which was comparable to that of 1; however, its cytotoxicity was also increased relative to 2. Our findings suggest that prodrugs of 4′-thioribonucleoside derivatives such as EICAR (1) represent an effective approach to developing potent biologically active compounds; however, the balance between antiviral activity and cytotoxicity remains to be addressed.

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