SUMMARY We have developed genetically encoded fluorescent sensors for reduced nicotinamide adenine dinucleotide (NADH), which manifest a large change in fluorescence upon NADH binding. We demonstrate the utility of these sensors in mammalian cells by monitoring the dynamic changes in NADH levels in subcellular organelles as affected by NADH transport, glucose metabolism, electron transport chain function, and redox environment, and we demonstrate the temporal separation of changes in mitochondrial and cytosolic NADH levels with perturbation. These results support the view that cytosolic NADH is sensitive to environmental changes, while mitochondria have a strong tendency to maintain physiological NADH homeostasis. These sensors provide a very good alternative to existing techniques that measure endogenous fluorescence of intracellular NAD(P)H and, owing to their superior sensitivity and specificity, allow for the selective monitoring of total cellular and compartmental responses of this essential cofactor.

A new chemosensor based on rhodamine B thiohydrazide is described. Chemosensor B was found to show a reversible dual chromo- and fluorogenic response toward Hg2+ in aqueous solution in a highly selective and sensitive manner. This was suggested to result from the coordination of Hg2+ at the N, S binding sites in B to open its spiro ring.

An alternative version of fluorescence correlation spectroscopy is presented, where the signal from a medium surrounding the particles of interest is analyzed, as opposed to a signal from the particles themselves. This allows for analysis of unlabeled particles and potentially of biomolecules. Here, the concept together with principal experiments on polystyrene beads of 100, 200, 400, and 800 nm diameter in an aqueous solution of alexa 488-fluorophores are presented. The use of photo detectors allowing higher photon fluxes, or of reduced detection volumes, should enable analysis of significantly smaller particles or even biomolecules.

Amphiphilic PNIPAM-b-PTPPC6MA block copolymers as promising photosensitizers for photodynamic therapy (PDT) constructed using porphyrin-containing monomers via RAFT polymerization.

Drug resistance is a primary obstacle that seriously reduces the therapy efficiency of most chemotherapeutic agents. To address this issue, the photochemical internalization (PCI) was employed to help the anticancer drug escape from lysosome and improve their translocation to the nucleus. A pH-sensitive porphyrin-based amphiphilic block copolymer (PEG-b-PCL-a-porphyrin) was synthesized, which was acted not only as a carrier for the delivery of DOX but also as a photosensitizer for PCI. PEG-b-PCL-a-porphyrin as a drug carrier exhibited a higher drug loading capacity, entrapment efficiency, and DOX release content. The PCI effect of PEG-b-PCL-a-porphyrin was studied by confocal laser scanning microscopy, and the results showed that most of DOX could be translocated into the nucleus for DOX-loaded PEG-b-PCL-a-porphyrin micelles. Moreover, the IC of pH-sensitive DOX-loaded PEG-b-PCL-a-porphyrin micelles was much lower than that of its counterpart without pH-responsiveness, DOX-loaded PEG-b-PCL-porphyrin micelles. Therefore, this drug delivery system based on pH-sensitive porphyrin-containing block copolymer would act as a potential vehicle for overcoming drug resistance in chemotherapy.

Room temperature phosphorescence emission was achieved by host–guest recognition between γ-cyclodextrin and a 4-bromo-1,8-naphthalic anhydride polymer, which can be controlled by the photo-isomerization of the azobenzene unit of the other polymer.

Fluorescence nanoscopy provides means to discern the finer details of protein localization and interaction in cells by offering an order of magnitude higher resolution than conventional optical imaging techniques. However, these super resolution techniques put higher demands on the optical system and the fluorescent probes, making multicolor fluorescence nanoscopy a challenging task. Here we present a new and simple procedure, which exploits the photostability and excitation spectra of dyes to increase the number of simultaneous recordable targets in STED nanoscopy. We use this procedure to demonstrate four-color STED imaging of platelets with e40 nm resolution and low crosstalk. Platelets can selectively store, sequester, and release a multitude of different proteins, in a manner specific for different physiological and disease states. By applying multicolor nanoscopy to study platelets, we can achieve spatial mapping of the protein organization with a high resolution for multiple proteins at the same time and in the same cell. This provides a means to identify specific platelet activation states for diagnostic purposes and to understand the underlying protein storage and release mechanisms. We studied the organization of the pro-and antiangiogenic proteins VEGF and PF-4, together with fibrinogen and filamentous actin, and found distinct features in their respective protein localization. Further, colocalization analysis revealed only minor overlap between the proteins VEGF and PF-4 indicating that they have separate storage and release mechanisms, corresponding well with their opposite roles as pro-and antiangiogenic proteins, respectively.

BackgroundPlatelets support cancer growth and spread making platelet proteins candidates in the search for biomarkers.MethodsTwo-dimensional (2D) gel electrophoresis, Partial Least Squares Discriminant Analysis (PLS-DA), Western blot, DigiWest.ResultsPLS-DA of platelet protein expression in 2D gels suggested differences between the International Federation of Gynaecology and Obstetrics (FIGO) stages III-IV of ovarian cancer, compared to benign adnexal lesions with a sensitivity of 96% and a specificity of 88%. A PLS-DA-based model correctly predicted 7 out of 8 cases of FIGO stages I-II of ovarian cancer after verification by western blot. Receiver-operator curve (ROC) analysis indicated a sensitivity of 83% and specificity of 76% at cut-off >0.5 (area under the curve (AUC) = 0.831, p < 0.0001) for detecting these cases. Validation on an independent set of samples by DigiWest with PLS-DA differentiated benign adnexal lesions and ovarian cancer, FIGO stages III-IV, with a sensitivity of 70% and a specificity of 83%.ConclusionWe identified a group of platelet protein biomarker candidates that can quantify the differential expression between ovarian cancer cases as compared to benign adnexal lesions.Electronic supplementary materialThe online version of this article (10.1186/s40364-018-0118-y) contains supplementary material, which is available to authorized users.