Trajectories: a framework for detecting temporal clinical event sequences from health data standardized to the Observational Medical Outcomes Partnership (OMOP) Common Data Model.

Objective: To develop a framework for identifying temporal clinical event trajectories from Observational Medical Outcomes Partnership-formatted observational healthcare data. Materials and Methods: A 4-step framework based on significant temporal event pair detection is described and implemented as an open-source R package. It is used on a population-based Estonian dataset to first replicate a large Danish population-based study and second, to conduct a disease trajectory detection study for type 2 diabetes patients in the Estonian and Dutch databases as an example. Results: As a proof of concept, we apply the methods in the Estonian database and provide a detailed breakdown of our findings. All Estonian population-based event pairs are shown. We compare the event pairs identified from Estonia to Danish and Dutch data and discuss the causes of the differences. The overlap in the results was only 2.4%, which highlights the need for running similar studies in different populations. Conclusions: For the first time, there is a complete software package for detecting disease trajectories in health data.

Intracellular dynamics of the Sigma-1 receptor observed with super-resolution imaging microscopy.

Sigma-1 receptor (Sig1R) is an endoplasmic reticulum (ER)-related membrane protein, that forms heteromers with other cellular proteins. As the mechanism of action of this chaperone protein remains unclear, the aim of the present study was to detect and analyze the intracellular dynamics of Sig1R in live cells using super-resolution imaging microscopy. For that, the Sig1R-yellow fluorescent protein conjugate (Sig1R-YFP) together with fluorescent markers of cell organelles were transfected into human ovarian adenocarcinoma (SK-OV-3) cells with BacMam technology. Sig1R-YFP was found to be located mainly in the nuclear envelope and in both tubular and vesicular structures of the ER but was not detected in the plasma membrane, even after activation of Sig1R with agonists. The super-resolution radial fluctuations approach (SRRF) performed with a highly inclined and laminated optical sheet (HILO) fluorescence microscope indicated substantial overlap of Sig1R-YFP spots with KDEL-mRFP, slight overlap with pmKate2-mito and no overlap with the markers of endosomes, peroxisomes, lysosomes, or caveolae. Activation of Sig1R with (+)-pentazocine caused a time-dependent decrease in the overlap between Sig1R-YFP and KDEL-mRFP, indicating that the activation of Sig1R decreases its colocalization with the marker of vesicular ER and does not cause comprehensive translocations of Sig1R in cells.

Deoxygenation Increases Photoluminescence Lifetime of Protein-Responsive Organic Probes with Triplet-Singlet Resonant Energy Transfer.

Cells and bodily fluids possess strong nanosecond-lifetime autofluorescence, therefore photoluminescent probes with microsecond-scale luminescence decay time would be useful for analysis of biological samples, as they allow the performance of measurements in time-resolved (TR) format in a time gate (time window) where the nonspecific background fluorescence has ceased. We have previously disclosed binding-responsive luminescent probes for protein kinases (PKs), ARC-Lum(Fluo) probes. High brightness of the probes is achieved through intramolecular Förster-type resonant energy transfer (FRET) from excited triplet state of a thiophene- or selenophene-comprising phosphor ((3)D*) to singlet acceptor dye ((1)A) leading to amplified emission from the dye. Here, we determined quantum yields (QYs) and oxygen sensitivity of separate phosphorescent donor and fluorescent acceptor and compared these with those of the corresponding ARC-Lum(Fluo) probes both in nonbound and PK-bound states. The microsecond-scale luminescence of free and of PK-bound probes was quenched with different efficiency by molecular oxygen and the luminescence intensity of the probes was substantially increased upon deoxygenation. The brightness of an ARC-Lum(Fluo) probe in PK-bound state was more than 50-fold higher than that of the phosphorescent donor alone. The findings of the study can be used for the construction of bright long-lifetime organic tandem probes.

Responsive microsecond-lifetime photoluminescent probes for analysis of protein kinases and their inhibitors.

Responsive ARC-Lum probes were used for measurement of the concentration of active protein kinases (PKs) and determination of affinity of inhibitors of PKs. ARC-Lum probes incorporate thiophene or a selenophene heterocycle and a fluorophore conjugated to the lysine residue in the peptide fragment. In the complex with a PK, ARC-Lum probes emit long-lifetime (microsecond-scale) luminescence at the emission wavelengths of the fluorescent label if the complex is illuminated at the excitation wavelength of the thiophene- or selenophene-containing phosphorescence donors. Bisubstrate ARC-Lum probes bind with sub-nanomolar affinity with several PKs of the AGC group. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).

Time-gated luminescence microscopy with responsive nonmetal probes for mapping activity of protein kinases in living cells.

A photoluminescence probe ARC-1185, possessing both high affinity towards basophilic protein kinases (PKs) and microsecond-scale luminescence lifetime when associated with a kinase, was used for the mapping of ARC-1185-PK complexes in living cells with time-gated luminescence microscopy.

Protein-induced long lifetime luminescence of nonmetal probes.

Time-resolved luminometry-based assays have great potential for measurements in complicated biological solutions and living cells as the measured signal can be easily distinguished from nanosecond lifetime background fluorescence of organic compounds and autofluorescence of cells. In the present study we discovered that binding of a thiophene- or a selenophene-containing heteroaromatic moiety (luminescence donor) to the purine-binding pocket of a protein kinase (PK) induces long lifetime photoluminescence signal that is largely intensified through efficient energy transfer to a fluorescent dye present in close proximity to the luminescence donor. The developed ARC-Lum probes possessing 19-266 µs luminescence lifetime when associated with the target kinase can be used for determination of activity of basophilic PKs, characterization of inhibitors of PKs, and as cAMP sensors. An ARC-Lum probe was also used for the determination of kinetic parameters of inhibitor binding to the catalytic subunit of protein kinase A (PKAc). Effective real-time monitoring of the activation of PKA by Forskolin and the displacement of an ARC-Lum probe from its complex with PKA by inhibitor H89 was performed in live cells. The discovered phenomenon, protein-induced long lifetime luminescence of aromatic probes is very likely to occur with all PKs and many other proteins.