Spatiotemporal Fluctuation Analysis: A Powerful Tool for the Future Nanoscopy of Molecular Processes

AbstractThe enormous wealth of information available today from optical microscopy measurements on living samples is often underexploited. We argue that spatiotemporal analysis of fluorescence fluctuations using multiple detection channels can enhance the performance of current nanoscopy methods and provide further insight into dynamic molecular processes of high biological relevance

Isothermal Microcalorimetry Detects the Presence of Persister Cells in a Staphylococcus aureus Biofilm After Vancomycin Treatment

Staphylococcus aureus biofilm plays a major role in implant-associated infections. Here, the susceptibility of biofilm S. aureus to daptomycin, fosfomycin, vancomycin, trimethoprim/sulfamethoxazole, linezolid, and rifampicin was investigated by isothermal microcalorimetry (IMC). Moreover, the persister status of cells isolated from S. aureus biofilm after treatment with vancomycin was also analyzed. S. aureus biofilm was tolerant to all the antibiotics tested [minimum biofilm bactericidal concentration (MBBC) 7> 256 mu g/ml], except to daptomycin [MBBC and minimum biofilm eradicating concentration (MBEC) = 32 mu g/ml] and rifampin (MBBC and MBEC = 128 mu g/ml). After the treatment of MRSA biofilm with 1024 mu g/ml vancomycin, similar to 5% cells survived, although metabolically inactive (persisters). Interestingly, IMC revealed that persister bacteria reverted to a normal-growing phenotype when inoculated into fresh medium without antibiotics. A staggered treatment of MRSA biofilm with vancomycin to kill all the metabolically active cells and daptomycin to kill persister cells eradicated the whole bacterial population. These results support the use in the clinical practice of a therapeutic regimen based on the use of two antibiotics to kill persister cells and eradicate MRSA biofilms. IMC represents a suitable technique to characterize in real-time the reversion from persister to metabolically-active cells

Isothermal Microcalorimetry Detects the Presence of Persister Cells in a Staphylococcus aureus Biofilm After Vancomycin Treatment

Staphylococcus aureus biofilm plays a major role in implant-associated infections. Here, the susceptibility of biofilm S. aureus to daptomycin, fosfomycin, vancomycin, trimethoprim/sulfamethoxazole, linezolid, and rifampicin was investigated by isothermal microcalorimetry (IMC). Moreover, the persister status of cells isolated from S. aureus biofilm after treatment with vancomycin was also analyzed. S. aureus biofilm was tolerant to all the antibiotics tested [minimum biofilm bactericidal concentration (MBBC) > 256 μg/ml], except to daptomycin [MBBC and minimum biofilm eradicating concentration (MBEC) = 32 μg/ml] and rifampin (MBBC and MBEC = 128 μg/ml). After the treatment of MRSA biofilm with 1024 μg/ml vancomycin, ∼5% cells survived, although metabolically inactive (persisters). Interestingly, IMC revealed that persister bacteria reverted to a normal-growing phenotype when inoculated into fresh medium without antibiotics. A staggered treatment of MRSA biofilm with vancomycin to kill all the metabolically active cells and daptomycin to kill persister cells eradicated the whole bacterial population. These results support the use in the clinical practice of a therapeutic regimen based on the use of two antibiotics to kill persister cells and eradicate MRSA biofilms. IMC represents a suitable technique to characterize in real-time the reversion from persister to metabolically-active cells

Precursor and mature NGF live tracking: one versus many at a time in the axons

The classical view of nerve growth factor (NGF) action in the nervous system is linked to its retrograde axonal transport. However, almost nothing is known on the trafficking properties of its unprocessed precursor proNGF, characterized by different and generally opposite biological functions with respect to its mature counterpart. Here we developed a strategy to fluorolabel both purified precursor and mature neurotrophins (NTs) with a controlled stoichiometry and insertion site. Using a single particle tracking approach, we characterized the axonal transport of proNGF versus mature NGF in living dorsal root ganglion neurons grown in compartmentalized microfluidic devices. We demonstrate that proNGF is retrogradely transported as NGF, but with a lower flux and a different distribution of numbers of neurotrophins per vesicle. Moreover, exploiting a dual-color labelling technique, we analysed the transport of both NT forms when simultaneously administered to the axon tips

Precursor and mature NGF live tracking: one versus many at a time in the axons

The classical view of nerve growth factor (NGF) action in the nervous system is linked to its retrograde axonal transport. However, almost nothing is known on the trafficking properties of its unprocessed precursor proNGF, characterized by different and generally opposite biological functions with respect to its mature counterpart. Here we developed a strategy to fluorolabel both purified precursor and mature neurotrophins (NTs) with a controlled stoichiometry and insertion site. Using a single particle tracking approach, we characterized the axonal transport of proNGF versus mature NGF in living dorsal root ganglion neurons grown in compartmentalized microfluidic devices. We demonstrate that proNGF is retrogradely transported as NGF, but with a lower flux and a different distribution of numbers of neurotrophins per vesicle. Moreover, exploiting a dual-color labelling technique, we analysed the transport of both NT forms when simultaneously administered to the axon tips