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During protein synthesis, transfer RNA and messenger RNA undergo coupled translocation through the ribosome's A, P and E sites, a process catalyzed by elongation factor EF-G. Viomycin blocks translocation on bacterial ribosomes and is believed to bind at the subunit interface. Using fluorescent resonance energy transfer and chemical footprinting, we show that viomycin traps the ribosome in an intermediate state of translocation. Changes in FRET efficiency show that viomycin causes relative movement of the two ribosomal subunits indistinguishable from that induced by binding of EF-G with GDPNP. Chemical probing experiments indicate that viomycin induces formation of a hybrid-state translocation intermediate. Thus, viomycin inhibits translation through a unique mechanism, locking ribosomes in the hybrid state; the EF-G-induced 'ratcheted' state observed by cryo-EM is identical to the hybrid state; and, since translation is viomycin sensitive, the hybrid state may be present in vivo.

Background/Aims: Senescent Ccl2^–/– mice develop cardinal features of human age-related macular degeneration (AMD). Loss-of-function single-nucleotide polymorphisms within CX3CR1 are associated with AMD. Methods: We generated Ccl2^–/–/Cx3cr1^–/– [double-knockout (DKO)] mice and evaluated the eyes using fundoscopy routine histology, immunochemistry, biochemistry and proteomics. Results: At 6 weeks old, all DKO mice developed AMD-like retinal lesions such as abnormal retinal pigment epithelium cells, drusen, photoreceptor atrophy and choroidal neovascularization, which progressed with age and reversed with high omega-3 long-chain polyunsaturated fatty acid diet. N-retinylidene-N-retinylethanolamine (A2E), a major lipofuscin fluorophore, illustrated by an emission peak at ∼600 nm, was significantly higher in DKO retinal pigment epithelium. Decreased ERp29 was found in the retina of DKO mice. Conclusion: A broad spectrum of AMD pathologies with early onset and high penetrance in these mice implicate certain chemokines, A2E and endoplasmic reticulum proteins in AMD pathogenesis.

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Purpose-To employ multiple modality imaging to described patients with type 2 idiopathic macular telangiectasia (IMT) at different disease severity stages so as to characterize and categorize disease progression through the full spectrum of disease phenotypes. Design-Observational case series.Methods-Twelve patients with type 2 IMT (22 eyes) with type 2 IMT examined with fundus photography, angiography, optical coherence tomography (OCT) imaging, fundus autofluorescence (FAF), and microperimetry (MP) testing in an institutional setting.Results-Eyes examined by multiple modality imaging were classified into five proposed categories (0-4): Category 0 (fellow) eyes were normal on all imaging modalities. Category 1 eyes had increased foveal autofluorescence on FAF imaging as the only imaging abnormality. Category 2 eyes had increased foveal autofluorescence together with funduscopic and angiographic features typical of type 2 IMT. Category 3 had additional evidence of foveal atrophy on OCT and while category 4 has all the above features plus clinically evident pigment clumping. FAF signal increased in intensity in the foveal region from category 0 through category 3, while category 4 eyes demonstrated a mixed pattern of increased and decreases FAF signal.Conclusions-The findings here outline a sequence of progressive changes seen with multiple imaging modalities in advancing stages of disease. Increases in foveal autofluorescence is an early anatomical change in type 2 IMT that may precede typical clinical and angiographic changes. Loss of macular pigment density in the fovea and a changing composition of flurophores in the retinal pigment epithelium may underlie these changes in FAF in the fundus.

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We present experiments that are convenient and educational for measuring fluorescence lifetimes with both time- and frequency-domain methods. The sample is ruby crystal, which has a lifetime of about 3.5 milliseconds, and is easy to use as a class-room demonstration. The experiments and methods of data analysis are used in the lab section of a class on optical spectroscopy, where we go through the theory and applications of fluorescence. Because the fluorescence decay time of ruby is in the millisecond region, the instrumentation for this experiment can be constructed easily and inexpensively compared to the nanosecond-resolved instrumentation required for most fluorescent compounds, which have nanosecond fluorescence lifetimes. The methods are applicable to other luminescent compounds with decay constants from microseconds and longer, such as transition metal and lanthanide complexes and phosphorescent samples. The experiments, which clearly demonstrate the theory and methods of measuring temporally resolved fluorescence, are instructive and demonstrate what the students have learned in the lectures without the distraction of highly sophisticated instrumentation.