Super-resolution optical imaging reveals the accumulation of SARS-CoV-2 nucleocapsid protein at viral replication organelles.

Herpesviruses are large and complex viruses that have a long history of coevolution with their host species. One important factor in the virus–host interaction is the alteration of intracellular morphology during viral replication with critical implications for viral assembly. However, the details of this remodeling event are not well understood, in part because insufficient tools are available to deconstruct this highly heterogeneous process. To provide an accurate and reliable method of investigating the spatiotemporal dynamics of virus-induced changes to cellular architecture, we constructed a dual-fluorescent reporter virus that enabled us to classify four distinct stages in the infection cycle of herpes simplex virus-1 at the single cell level. This timestamping method can accurately track the infection cycle across a wide range of multiplicities of infection. We used high-resolution fluorescence microscopy analysis of cellular structures in live and fixed cells in concert with our reporter virus to generate a detailed and chronological overview of the spatial and temporal reorganization during viral replication. The highly orchestrated and striking relocation of many organelles around the compartments of secondary envelopment during transition from early to late gene expression suggests that the reshaping of these compartments is essential for virus assembly. We furthermore find that accumulation of HSV-1 capsids in the cytoplasm is accompanied by fragmentation of the Golgi apparatus with potential impact on the late steps of viral assembly. We anticipate that in the future similar tools can be systematically applied for the systems-level analysis of intracellular morphology during replication of other viruses.

In this work, we report the synthesis of [Ru(phen) 3 2+ ]based complexes and their use as photosensitizers for photodynamic therapy (PDT), a treatment of pathological conditions based on the photoactivation of bioactive compounds, which are not harmful in the absence of light irradiation. Of these complexes, Ru-PhenISA and Ru-PhenAN are polymer conjugates containing less than 5%, (on a molar basis), photoactive units. Their performance is compared with that of a small [Ru(phen) 3 2+ ] compound, [Ru(phen) 2 BAP](OTf) 2 (BAP = 4-(4′-aminobutyl)-1,10-phenanthroline, OTf = triflate anion), used as a model of the photoactive units. The polymer ligands, PhenISA and PhenAN, are polyamidoamines with different acid−base properties. At physiological pH, the former is zwitterionic, the latter moderately cationic, and both intrinsically cytocompatible. The photophysical characterizations show that the complexation to macromolecules does not hamper the Ru(phen) 3 2+ ability to generate toxic singlet oxygen upon irradiation, and phosphorescence lifetimes and quantum yields are similar in all cases. All three compounds are internalized by HeLa cells and can induce cell death upon visible light irradiation. However, their relative PDT efficiency is different: the zwitterionic PhenISA endowed with the Ru-complex lowers the PDT efficiency of the free complex, while conversely, the cationic PhenAN boosts it. Flow cytometry demonstrates that the uptake efficiency of the three agents reflects the observed differences in PDT efficacy. Additionally, intracellular localization studies show that while [Ru(phen) 2 BAP](OTf) 2 remains confined in vesicular structures, Ru-PhenISA localization is hard to determine due to the very low uptake efficiency. Very interestingly, instead, the cationic Ru-PhenAN accumulates inside the nucleus in all treated cells. Overall, the results indicate that the complexation of [Ru(phen) 2 BAP](OTf) 2 with a cationic polyamidoamine to give the Ru-PhenAN complex is an excellent strategy to increase the Ru-complex cell uptake and, additionally, to achieve accumulation at the nuclear level. These unique features together make this compound an excellent photosensitizer with very high PDT efficiency.

wileyonlinelibrary.com COMMUNICATION30 °C and every 6 h at 20 °C. Assuming an ambient temperature in the range of 20-25 °C, a suitable label should provide a clear signal to be read by the naked eye in around 3 h. [ 12 ] We here describe a new class of irreversible thermochromic molecular materials whose chemical transformation-when deposited as thin fi lms-from a colorless state to a strongly colored one depends upon temperature, chemical substitution, and substrate. The selection of the appropriate active molecule/ substrate combination provides sensitivity to different targeted time/temperature regimes, coherent with the sensitivity limits just described.The key process providing the required irreversible color change is connected with the "latent pigment" approach. [ 14 ] This process enables to induce the reversible transformation of organic pigments-like diketopyrrolopyrroles (as the red 254 pigment shown in Scheme S1 in the Supporting Information) [ 15 ] and quinacridones [ 16 ] -into soluble dyes by means of the protection of its hydrogen bond forming functionalities. As this is usually done by reaction of the pigment with ditert-butyl dicarbonate, the resulting tert-butylcarbonate ( t BOC) protection is both thermal and acid labile, with the evolution of CO 2 and isobutene (Scheme S1, Supporting Information). [ 17 ] Importantly, note that due to the involved (minor) change in the optical gap and (more importantly) aggregation state, the latent pigments are in general irreversible thermochromic materials, as we have recently shown in the context of organic solar cells. [ 15 ] Once applied to a very peculiar class of pigments-the 1,3-squaraines [ 18 ] shown in Scheme 1 , the optical gap variation associated with the latent pigment process becomes extreme, turning the blue pigment into a colorless molecule. This reaction provides the kind of dramatic color change required for smart labels directly readable by the naked eye and it is thermally irreversible (see below), thus ensuring a reliable reading by the customer. In details, Scheme 1 shows that the treatment of squaraines 1 and 2 with di-tert-butyl dicarbonate and 4-N , N -dimethylaminopyridine (DMAP) leads to the formation of betaines 3 and 4 . Since the addition of DMAP to the squarylium double bond breaks the typical cyanine conjugation of the dye, said betaines have no visible absorption. It should be noted, though, that under analogous experimental conditions, DMAP alone did not react with squaraines. Possibly, such unusual reactivity is a consequence of the severe twisting induced by the introduction of the four t BOC bulky groups in close proximity to the squarylium core. Such distortion makes the squarylium double bond more reactive toward the nucleophile addition. The pristine squarylium dye can be quantitatively recovered by thermal treatment or by addition of trifl uoroacetic acid, according to the well know acid catalyzed cleavage of t BOC groups.Food safety has always been a major concern for mankind. The wide availability of fresh perishab...