Bioorthogonal fluorescent labels: a review on combined forces

This review ventures to summarize the latest developments in bioorthogonal fluorescent imaging labels with a special focus on bioimaging applications. We briefly summarize the most preferred means of bioorthogonal tagging schemes for the labeling of specific biomolecular structures. The review is structured by the type of the fluorescent labels that can address the problems that most commonly compromise fluorescent imaging techniques, i.e. the autofluorescence of biomolecules, the background fluorescence of unreacted reagents, and photobleaching. Thus, we present (i) far- red/near-infra-red emitting dyes, (ii) fluorogenic scaffolds, and (iii) nanoparticle-based signaling platforms

Microglia control the spread of neurotropic virus infection via P2Y12 signalling and recruit monocytes through P2Y12-independent mechanisms

Neurotropic herpesviruses can establish lifelong infection in humans and contribute to severe diseases including encephalitis and neurodegeneration. However, the mechanisms through which the brain's immune system recognizes and controls viral infections propagating across synaptically linked neuronal circuits have remained unclear. Using a well-established model of alphaherpesvirus infection that reaches the brain exclusively via retrograde transsynaptic spread from the periphery, and in vivo two-photon imaging combined with high resolution microscopy, we show that microglia are recruited to and isolate infected neurons within hours. Selective elimination of microglia results in a marked increase in the spread of infection and egress of viral particles into the brain parenchyma, which are associated with diverse neurological symptoms. Microglia recruitment and clearance of infected cells require cell-autonomous P2Y12 signalling in microglia, triggered by nucleotides released from affected neurons. In turn, we identify microglia as key contributors to monocyte recruitment into the inflamed brain, which process is largely independent of P2Y12. P2Y12-positive microglia are also recruited to infected neurons in the human brain during viral encephalitis and both microglial responses and leukocyte numbers correlate with the severity of infection. Thus, our data identify a key role for microglial P2Y12 in defence against neurotropic viruses, whilst P2Y12-independent actions of microglia may contribute to neuroinflammation by facilitating monocyte recruitment to the sites of infection

Microglia control the spread of neurotropic virus infection via P2Y12 signalling and recruit monocytes through P2Y12-independent mechanisms

Neurotropic herpesviruses can establish lifelong infection in humans and contribute to severe diseases including encephalitis and neurodegeneration. However, the mechanisms through which the brain's immune system recognizes and controls viral infections propagating across synaptically linked neuronal circuits have remained unclear. Using a well-established model of alphaherpesvirus infection that reaches the brain exclusively via retrograde transsynaptic spread from the periphery, and in vivo two-photon imaging combined with high resolution microscopy, we show that microglia are recruited to and isolate infected neurons within hours. Selective elimination of microglia results in a marked increase in the spread of infection and egress of viral particles into the brain parenchyma, which are associated with diverse neurological symptoms. Microglia recruitment and clearance of infected cells require cell-autonomous P2Y12 signalling in microglia, triggered by nucleotides released from affected neurons. In turn, we identify microglia as key contributors to monocyte recruitment into the inflamed brain, which process is largely independent of P2Y12. P2Y12-positive microglia are also recruited to infected neurons in the human brain during viral encephalitis and both microglial responses and leukocyte numbers correlate with the severity of infection. Thus, our data identify a key role for microglial P2Y12 in defence against neurotropic viruses, whilst P2Y12-independent actions of microglia may contribute to neuroinflammation by facilitating monocyte recruitment to the sites of infection

NIR mega-Stokes fluorophores for bioorthogonal labeling and energy transfer systems - An efficient quencher for daunomycin.

A set of new, azide and alkyne bearing lepidinium-based fluorophores were synthesized for bioorthogonal labeling schemes. These fluorescent dyes all show large Stokes-shifts with emission maxima in the near-infra-red (NIR) region of the electromagnetic spectrum. The applicability of these dyes in the construction of energy transfer systems was tested using one of these new fluorescent tags and daunomycin (Dau), an anticancer drug with fluorescent features. These daunomycin conjugates are the very first examples for fluorescently modulated constructs of this anticancer agent. The dually labeled architectures proved that the applied fluorescent dye can be utilized as an efficient quencher for daunomycin. Enzymatic cleavage of a dually labeled enzyme substrate resulted in full recovery of the fluorescence of daunomycin. Such fluorescently modulated Dau-conjugates can provide useful information for the action-mechanism of Dau-regulated cell death processes

A Genetically Encoded Isonitrile Lysine for Orthogonal Bioorthogonal Labeling Schemes

Bioorthogonal click-reactions represent ideal means for labeling biomolecules selectively and specifically with suitable small synthetic dyes. Genetic code expansion (GCE) technology enables efficient site-selective installation of bioorthogonal handles onto proteins of interest (POIs). Incorporation of bioorthogonalized non-canonical amino acids is a minimally perturbing means of enabling the study of proteins in their native environment. The growing demand for the multiple modification of POIs has triggered the quest for developing orthogonal bioorthogonal reactions that allow simultaneous modification of biomolecules. The recently reported bioorthogonal [4 + 1] cycloaddition reaction of bulky tetrazines and sterically demanding isonitriles has prompted us to develop a non-canonical amino acid (ncAA) bearing a suitable isonitrile function. Herein we disclose the synthesis and genetic incorporation of this ncAA together with studies aiming at assessing the mutual orthogonality between its reaction with bulky tetrazines and the inverse electron demand Diels–Alder (IEDDA) reaction of bicyclononyne (BCN) and tetrazine. Results showed that the new ncAA, bulky-isonitrile-carbamate-lysine (BICK) is efficiently and specifically incorporated into proteins by genetic code expansion, and despite the slow [4 + 1] cycloaddition, enables the labeling of outer membrane receptors such as insulin receptor (IR) with a membrane-impermeable dye. Furthermore, double labeling of protein structures in live and fixed mammalian cells was achieved using the mutually orthogonal bioorthogonal IEDDA and [4 + 1] cycloaddition reaction pair, by introducing BICK through GCE and BCN through a HaloTag technique